Detection of canine microalbuminuria using semiquantitative test strips designed for use with human urine

Background — Commercial testing for microalbuminuria in human urine is often performed with point-of-care semiquantitative test strips followed by quantitative testing when indicated. An ELISA that quantifies canine urine albumin concentration has been developed, but semiquantitative test strips for use in the dog are not available.
Objective — The purpose of this study was to prospectively determine the concordance of canine urine albumin concentrations measured by a commercial human test strip and by ELISA.
Methods — Urine samples were obtained from 67 dogs evaluated for a variety of clinical conditions. Dipstick urinalyses were performed on all samples; clinician discretion determined method of urine collection and performance of urine sediment examination and/or urine culture. Urine albumin concentration was determined using test strips (Clinitek Microalbumin, Bayer Corporation, Elkhart, Ind, USA), and results were compared with those obtained by ELISA.
Results — The Clinitek strips correctly determined albumin concentration in 42 of 67 (63%) urine samples tested. Concordance was lowest (48%) for dogs with microalbuminuria (10–300 μg/mL by ELISA). Clinitek strip sensitivity and specificity for correct identification of microalbuminuria were 48% and 75%, respectively. Concordance was lower in dogs with urinary tract infection or hematuria and in samples collected by catheterization. Sensitivity and specificity for correct identification of microalbuminuria after exclusion of dogs with urinary tract infection or hematuria were 59% and 83%, respectively.
Conclusion — These results suggest that the Clinitek strips lack sufficient concordance with results obtained by ELISA to be reliable screening tests for microalbuminuria in the dog. A reliable semiquantitative point-of-care test for canine urine albumin concentrations below those detected by standard urine dipsticks is still needed.     

Assessment of five portable blood glucose meters, a point-of-care analyzer, and color test strips for measuring blood glucose concentration in dogs

OBJECTIVE: To compare blood glucose concentrations obtained using a point-of-care (POC) analyzer, 5 portable blood glucose meters (PBGM), and a color reagent test strip with concentrations obtained using a reference method, and to compare glucose concentrations obtained using fresh blood samples in the PBGM with concentrations obtained using blood anticoagulated with lithium heparin. DESIGN: Case series. SAMPLE POPULATION: 110 blood samples from 34 dogs; glucose concentration of the samples ranged from 41 to 596 mg/dl. PROCEDURE: Logistic regression was used to compare blood glucose concentrations obtained with the various devices with reference method concentrations. Ease of use was evaluated subjectively. Percentage of times a clinical decision would have been altered if results of each of these methods had been used, rather than results of the reference method, was calculated. RESULTS: For 3 of the PBGM, blood glucose concentrations obtained with fresh blood were not significantly different from concentrations obtained with blood samples anticoagulated with lithium heparin. None of the devices provided results statistically equivalent to results of the reference method, but the POC analyzer was more accurate than the others. For some samples, reliance on results of the PBGM or the color test strip would have resulted in erroneous clinical decisions. CONCLUSIONS AND CLINICAL RELEVANCE: Although commercially available PBGM and color test strips provided blood glucose concentrations reasonably close to those obtained with reference methods, some devices were more accurate than others. Use of results from these devices could lead to erroneous clinical decisions in some cases.     

Effects of dietary protein and stage of lactation on the haematology and erythrocyte enzymes activities of high-producing dairy cattle.

Blood samples were collected monthly over a nine-month period from 19 high-producing Holstein-Friesian dairy cows. Dry cows on the lowest (13 per cent) protein ration had the highest mean values for packed cell volume (PCV), haemoglobin (Hb) and red blood cells (RBC). Among the lactating cows, the group on the 13 per cent protein diet had the highest mean PCV, Hb and RBC values. Other constituents were not affected significantly by dietary protein levels. Packed cell volume, RBC, Hb, serum iron (SI), iron binding capacity (IBC) and serum albumin concentrations decreased early in lactation and rose to pre-lactation levels by mid-lactation. PCV and Hb concentrations remained low for periods up to four months. RBC count was lowest in the second month while albumin concentration was lowest in the first month and remained low up to the second month. IBC was lowest in the first month of lactation while SI concentrations were lowest in the third month. There were no significant variations in the activities of erythrocyte glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD) and reduced glutathione (GSH). The 13 per cent protein ration had no anaemia-inducing effect on the cows.     

Elevated concentrations in serum immunoglobulins due to infection by ovine progressive pneumonia virus.

Sixty-seven serum samples were obtained from 2 sheep flocks. Agar gel immunodiffusion (AGID) was used to separate progressive pneumonia virus (PPV)-infected sheep from noninfected sheep by the presence of precipitating antibodies. Immunoglobulin (Ig), total protein, and albumin concentrations were then measured from all 67 sera to determine whether differences existed between PPV-infected and non-infected sheep. A significant difference (P less than 0.0005) was found in both total protein and Ig concentration between PPV-infected and noninfected sheep. This corresponding difference was absent in albumin measurements. The significant differences (P less than 0.0005) in Ig and total protein concentrations were then used to evaluate a field test for diagnosing progressive pneumonia. The possibility of using either total protein or Ig concentrations as a field test was found to be highly unlikely due to variation in individual values.     

Validation of a human immunoturbidimetric assay to measure canine albumin in urine and cerebrospinal fluid.

The aim of this study was to validate an automated immunoturbidimetric assay used to quantify human albumin in urine and to accurately measure canine albumin concentrations in both urine and cerebrospinal fluid. The partial homology existing between human and canine albumin limited the accuracy of the human assays in measuring canine albumin without method modifications. Thus, the assay was modified by calibrating the analyzer with calibrators made in the laboratory containing known concentrations of canine albumin. To prepare the set of calibrators, the albumin concentration of pooled sera of healthy dogs was assessed in 5 replicates using the BromocresolGreen assay. Pooled samples were aliquoted and serially diluted to obtain the expected concentrations of albumin (0.5, 1, 5, 13, and 30 mg/dl) for establishing the canine calibration curve. Thereafter, the performance was assessed by analyzing canine urine and CSF The modified assay accurately quantified canine albumin in both specimens, as indicated by the following. Intra- and interassay variability was 0.92% and 2.74%, respectively; recovery was 99.66% and 99.07% in urine and 105.02% in CSF No interference was detected when hemolysate and glucose were added to urine. The test was linear within the verified range (0-225 mg/dl). These results demonstrate that the modified human albumin immunoturbidimetric assay can be a useful tool in the veterinary diagnostic laboratory. It is accurate and tends itself to automatization on chemistry analyzers.     

Comparison of methods for depletion of albumin and IgG from equine serum

Background: Disease‐specific biomarkers hold diagnostic promise in both human and veterinary medicine, but serum biomarkers in low concentrations may be masked by the presence of abundant proteins, mostly albumin and IgG. Methods to deplete albumin and IgG exist, but efficacy of these methods for depleting equine serum of these proteins has not been established. Objective: The aim of this study was to determine if albumin and IgG could be depleted from equine serum using several commercially available kits and procedures. Methods: One‐dimensional gel electrophoresis followed by densitometry was used to determine percent of albumin, IgG, and both in pooled serum from 3 horses before and after application of 7 depletion methods. Repeatability was determined by applying the 2 best methods to serum samples from 6 grade horses. Results: For pooled serum, depletion rates varied from 35–90% for albumin and 0–94% for IgG. In the repeatability study, the ProteoExtract method combined with protein G Sepharose beads to remove additional IgG provided the best overall performance with 66% albumin depletion and 100% IgG depletion. A protocol using protein G Sepharose beads to remove IgG followed by ethanol precipitation of nonalbumin proteins with albumin remaining in the supernatant was the second most effective, with 85% albumin depletion and 55% IgG depletion. Although a multiprotein immunodepletion column effectively removed 90% of the albumin, the method was ineffective at removing IgG. Conclusion: Albumin and IgG removal kits optimized for human use have variable efficacy for equine serum. Combined use of the ProteoExtract kit and manual incubation with protein G Sepharose beads provided the most effective depletion.     

Separation of serum glycated proteins by agarose gel electrophoresis and nitroblue tetrazolium staining in diabetic and normal cats

BACKGROUND: The total glycated protein (fructosamine) concentration in serum consists mainly of glycated albumin and lipoproteins. Measurement of fructosamine is used to diagnose and monitor diabetes mellitus in cats. OBJECTIVE: The aims of this study were to measure glycated proteins in diabetic and healthy (nondiabetic) cats using a semiquantitative technique and to determine whether measurement of any of the fractions of glycated protein could be potentially advantageous for the diagnosis and monitoring of diabetic cats. METHODS: Serum samples from 6 cats with diabetes mellitus and 10 clinically healthy adult cats were assayed for total glycated protein using a nitroblue tetrazolium (NBT) fructosamine assay. Serum proteins were separated by agarose gel electrophoresis and stained with NBT to identify individual glycated proteins within the bands. Gels were scanned by densitometry at 525 nm and the glycated protein content was calculated with reference to the total glycated protein content of the sample. RESULTS: Diabetic cats with increased total fructosamine concentrations had higher concentrations of glycated albumin and glycated alpha- and beta-lipoproteins compared with healthy cats. The concentration of glycated proteins in each of the fractions had a positive linear association with the total glycated protein content of serum, but there was large variation in the relative contributions of the 3 protein fractions to the total glycated protein concentration. CONCLUSIONS: Based on the results of this study, measurement of individual glycated fractions does not seem to offer any potential diagnostic advantage over measurement of total glycated protein (fructosamine) concentration alone. In some diabetic and healthy cats, glycated lipoproteins formed the major part of the total glycated protein, whereas in other cats albumin was the major contributor.     

Comparison of semiquantitative test strips,urine protein electrophoresis,and an immunoturbidimetric assay for measuring microalbuminuria in dogs

Background: The presence of albumin in urine, even in small amounts, is always abnormal and usually reflects kidney dysfunction. Different techniques are commercially available for the measurement of microalbuminuria in dogs. Objectives: The purpose of this study was to compare the accuracy of semiquantitative test strips, urine protein electrophoresis, and a validated immunoturbidimetric assay in the measurement of microalbuminuria in dogs. Methods: Urine samples were collected from 307 dogs presented to The Queen's Veterinary School Hospital, University of Cambridge, for a variety of clinical conditions. Urine was collected by midstream free catch (193/307, 63%), cystocentesis (89/307, 29%), or catheterization (25/307, 8%). Routine urinalysis was performed on all samples. Albumin was measured by using semiquantitative test strips, by agarose gel electrophoresis, and by an automated immunoturbidimetric assay designed for human samples (considered as the gold standard). The latter was validated using a purified canine albumin standard. Results: The immunoturbidimetric assay had within‐assay and between‐assay coefficients of variation (CV) of 1.3% and 5.0%, respectively, overall recovery of 97.1%, and high linearity (r=.985). Of the samples with measurable albumin (>1.4 mg/L) by the immunoturbidimetric assay, 57/195 (29%) were negative for albumin using the semiquantitative test strips and 138/195 (71%) were positive. Urine protein electrophoresis (UPE) and immunoturbidimetric results had a concordance CV of 86%. Conclusions: UPE and semiquantitative test strips are less accurate than the automated immunoturbidimetric method for the measurement of albumin in canine urine.     

Agarose gel electrophoresis of cerebrospinal fluid proteins of dogs after sample concentration using a membrane microconcentrator technique

BACKGROUND: Cerebrospinal fluid (CSF) is produced in the cerebral ventricles through ultrafiltration of plasma and active transport mechanisms. Evaluation of proteins in CSF may provide important information about the production of immunoglobulins within the central nervous system as well as possible disturbances in the blood-brain barrier. OBJECTIVE: The objective of this study was to measure the concentration and fractions of protein in CSF samples using a membrane microconcentrator technique followed by electrophoresis, and to compare the protein fractions obtained with those in serum. METHODS: CSF samples from 3 healthy dogs and 3 dogs with canine distemper virus infection were concentrated using a membrane microconcentrator having a 0.5 to 30,000 d nominal molecular weight limit (Ultrafree, Millipore, Billerica, MA, USA). Protein concentration was determined before and after concentration. Agarose gel electrophoresis was done on concentrated CSF samples, serum, and serial dilutions of one of the CSF samples. RESULTS: Electrophoretic bands were clearly identified in densitometer tracings in CSF samples with protein concentrations as low as 1.3 g/dL. The higher CSF protein concentration in dogs with distemper was mainly the result of increased albumin concentration. CONCLUSION: The microconcentrating method used in this study enables characterization of the main protein fractions in CSF by routine electrophoresis and may be useful for interpreting the underlying cause of changes in CSF protein concentrations.     

Pathophysiological studies on Dicrocoelium dendriticum infection in sheep

Red cell kinetics and plasma protein metabolism were investigated in two experiments using 20 adult sheep naturally infected with Dicrocoelium dendriticum, but free of other liver and gastrointestinal helminths. In the first experiment, where groups of animals with low to high Dicrocoelium burdens were injected with 51Cr-labelled red cells and 125I-labelled albumin, the results indicated that there were no significant differences in the turnover rate of labelled red cells or albumin between any of the groups. In the second experiment, two groups of sheep with low and high worm burdens were studied using the same radioisotope tracers; in addition, 59Fe-citrate was used to assess red cell iron incorporation rates in the two groups. Although the red cell half-lives of the infected sheep were just significantly longer, both were within normal limits and the difference was attributed to random variation within the two small groups of sheep. No significant differences were found in the other parameters. It was concluded that burdens of up to 4000 D. dendriticum do not cause significant blood or plasma protein loss in sheep.     

Comparison of four methods for determination of total protein concentrations in pleural and peritoneal fluid from dogs

OBJECTIVE: To compare 4 techniques for determination of total protein concentrations in peritoneal and pleural effusions from dogs. SAMPLE POPULATION: 23 peritoneal and 12 pleural fluid samples from 35 dogs with various abnormalities. PROCEDURE: Samples were collected into tubes containing EDTA, centrifuged, and stored at -20 C until total protein concentrations were assessed. Protein concentration in each sample was determined by use of urine test strips, refractometry, and Bradford and biuret techniques. Accuracy of each method was determined, using dilutions of human control sera. RESULTS: There was good correlation among results of all quantitative procedures. Results of the biuret technique were more accurate than results of the Bradford assay. Refractometry underestimated protein concentration in samples with < 20 g of protein/L. Results of urine test strips correctly classified effusion samples into 2 groups on the basis of total protein concentrations less than or greater than 20 g/L. CONCLUSIONS AND CLINICAL RELEVANCE: Results of any of these 4 techniques can be used to rapidly and efficiently differentiate peritoneal and pleural fluid from dogs into transudates and exudates on the basis of total protein concentration less than or greater than 20 g/L, respectively.     

Assessment of the reliability of plasma electrophoresis in birds

OBJECTIVE: To determine the reliability of plasma electrophoresis (EPH) in psittacine birds. ANIMALS: 93 psittacine birds. PROCEDURE: Jugular venipuncture was performed on 93 awake psittacine birds. The plasma was centrifuged, separated, aliquoted into duplicate samples, frozen, and sent to 2 commercial laboratories that routinely perform avian EPH. Samples from 51 birds were sent to laboratory A, and samples from 42 birds were sent to laboratory B. The reliability of EPH results within each laboratory was assessed, but not between laboratories. To determine the reliability (agreement between duplicate samples) of total protein, albumin, prealbumin, alpha1-, alpha2-, beta-, and gamma-globulin concentrations, the intraclass correlation coefficient (r(i)) was calculated. RESULTS: Both laboratories had excellent agreement between samples for measurement of total protein concentration and only good agreement for albumin concentration. Except for the prealbumin concentration measured at laboratory B, both laboratories had poor agreement for all other values of the EPH. CONCLUSIONS AND CLINICAL RELEVANCE: These data indicate that plasma EPH for measuring prealbumin, alpha1-, alpha2-, beta-, and gamma-globulin concentrations may not be a reliable tool for assessing avian health. Small amounts of these proteins in birds plus human variation in reading the EPH curves may lead to variable results. Avian veterinarians should cautiously interpret results from plasma EPH assays for these protein fractions.     

Evaluation of nephelometry for albumin measurement in serum and cerebrospinal fluid: experiences with an indwelling subarachnoidal catheter system for repetitive cerebrospinal fluid collection in horses.

The measurement of albumin concentrations in cerebrospinal fluid (CSF) and serum for albumin quotient (AQ) calculations in normal horses was performed by 2 methods: 1) total protein measurement, followed by electrophoresis of the samples to obtain an albumin percentage; and 2) albumin immunoprecipitation quantitated by nephelometry. The results of both methods correlated well, and nephelometry was chosen to determine the albumin concentrations in CSF samples obtained from an indwelling subarachnoidal catheter for daily sampling. Because the use of an indwelling catheter to collect repetitive CSF samples is a novel technique, routine cytological CSF analysis was performed along with daily clinical evaluation to ascertain the well-being of the horses. The catheters were placed in 2 horses for periods of 14 and 17 days. One horse exhibited pleocytosis on cytological evaluation of CSF on 2 occasions for a 1-2-day duration; however, the AQ showed a significant increase on only 1 occasion. The other horse had a normal cell count in CSF but showed 2 sudden changes in the AQ value; however, these values remained within the 95% confidence interval for AQ in horses. Albumin quotient values of the second horse were consistently below the lower range of the confidence interval. Results from this study indicate that nephelometry can be used for albumin determination in serum and CSF samples from horses. Furthermore, an indwelling subarachnoidal catheter system can provide serial CSF samples in horses, thus obviating the need for repetitive centesis for serial CSF sampling.     

Assessment of a point-of-care biochemical analyzer and comparison with a commercial laboratory for the measurement of total protein and albumin concentrations in psittacines

OBJECTIVE: To determine agreement for total protein (TP) and albumin concentrations measured by a point-of-care biochemical analyzer in heparinized whole blood and plasma samples obtained from psittacines and compare results with those from a commercial laboratory. SAMPLE POPULATION: Hematologic samples from 92 healthy birds. PROCEDURES: Duplicate samples of heparinized whole blood and plasma were obtained. A point-of-care biochemical analyzer was used to determine TP and albumin concentrations. To assess precision, intraclass correlation coefficient (r(i)) and Bland-Altman measures of agreement were used. These results were compared by use of Bland-Altman plots with those obtained from a commercial laboratory that used a biuret method for TP concentration and electrophoresis for albumin concentration. RESULTS: For the analyzer, there was excellent agreement (r(i) = 0.91) between heparinized whole blood and plasma samples for TP and albumin concentrations. Relative error was 0.9% for TP and 0.7% for albumin. Analyzer results correlated well with commercial laboratory results, with a downward bias of 0.6 for TP and 0.3 for albumin. CONCLUSIONS AND CLINICAL RELEVANCE: The analyzer had excellent precision for analysis of heparinized whole blood or plasma samples for TP or albumin concentrations; analyzer values had good agreement with those from a commercial laboratory. The analyzer could be a valid method to measure plasma TP concentrations and provide point-of-care testing in apparently healthy parrots. Biochemical analyzer results for plasma albumin concentration were not validated by results from a commercial laboratory, so conclusions cannot be drawn regarding use of the analyzer in measurement of albumin concentrations in psittacines.     

Evaluation of a Rapid Reagent Strip Test for Blood Glucose Determination in Diarrheic Calves

Sixty-three blood samples from 10 diarrheic calves were tested for glucose concentration by two methods. Plasma glucose concentration was measured by the conventional glucose-6-phosphate dehydrogenase method in the clinical laboratory, and the results compared to those obtained using a rapid reagent strip test for blood glucose concentration measurement. The rapid reagent strip test result could not be used to make an accurate prediction of the actual plasma glucose concentration as determined by the conventional method, due to the wide variability in actual plasma glucose concentrations corresponding to each rapid test result.     

Evaluation of a rapid reagent strip test for blood glucose determination in diarrheic calves

Sixty-three blood samples from 10 diarrheic calves were tested for glucose concentration by two methods. Plasma glucose concentration was measured by the conventional glucose-6-phosphate dehydrogenase method in the clinical laboratory, and the results compared to those obtained using a rapid reagent strip test for blood glucose concentration measurement. The rapid reagent strip test result could not be used to make an accurate prediction of the actual plasma glucose concentration as determined by the conventional method, due to the wide variability in actual plasma glucose concentrations corresponding to each rapid test result.     

Diagnosis of larval cyathostominosis in horses in Belgium.

Between October 1996 and May 1997, 94 horses which were suspected of being infected with strongyles were examined clinically, and samples of faeces were examined for strongyle eggs and cyathostome larvae (L4) and adults. Blood samples were monitored for total protein, albumin and beta-globulins. In 28 of the horses (30 per cent) cyathostome L4 and adults were detected in the faeces, and were significantly associated with the horses' condition, the occurrence of diarrhoea, with lower concentrations of total protein and albumin, and with higher percentages of beta-globulin. Thirty-four of the horses (36 per cent) were excreting strongyle eggs, but in these animals the associations were with high concentrations of total protein and albumin, and lower percentages of beta-globulin. The results showed that during the winter, a horse in poor condition which has diarrhoea, an albumin concentration less than 20 g/litre, and a ratio of albumin:globulin less than 0.7 is very likely to be infected with L4 and adult stages of cyathostomes.     

Serum protein electrophoresis in retired racing Greyhounds

BACKGROUND: Retired racing Greyhounds are becoming common as pets. Because of their unique physiology, results of routine laboratory tests are frequently outside the reference interval for dogs. Compared with other breeds, Greyhounds have low serum protein concentrations, but the concentrations of different serum protein fractions have not been reported. OBJECTIVES: Our objectives were to evaluate the results of serum protein electrophoresis (SPE) in healthy, retired racing Greyhounds and compare them with a control group of age- and gender-matched non-Greyhound dogs. METHODS: Agarose gel electrophoresis was done using a standard method; the gels were stained with amido black and scanned with a Cliniscan 2 densitometer (Helena Laboratories, Beaumont, TX, USA). Protein fractions were identified by visual inspection of the electrophoretogram. A Student's t-test assuming equal variances was used to compare the concentration of the different fractions between groups. RESULTS: The concentrations of total protein, total globulins, and alpha-1-, alpha-2-, beta-1-, and beta-2-globulins were significantly lower and the albumin to globulin (A:G) ratio was significantly higher in Greyhounds than in non-Greyhound dogs (P < .05). There was no significant difference in albumin or gamma-globulin concentrations. CONCLUSIONS: Low serum protein concentrations in Greyhounds are the result of low concentrations of a- and b-globulins. These results should be kept in mind when evaluating both healthy and sick Greyhounds. Additional studies are needed to identify the individual proteins associated with low alpha- and beta-globulin concentrations in Greyhounds.     

Study of the effect of total serum protein and albumin concentrations on canine fructosamine concentration.

The relationship among serum fructosamine concentration and total serum protein and albumin concentrations were evaluated in healthy and sick dogs (diabetics and dogs with insulinoma were not included). Fructosamine was determined using a commercial colorimetric nitroblue tetrazolium method applied to the Technicon RA-500 (Bayer). Serum fructosamine concentration was not correlated to total protein in normoproteinemic (r = 0.03) and hyperproteinemic dogs (r = 0.29), but there was a high correlation (r = 0.73) in hypoproteinemic dogs. Similar comparison between serum fructosamine and albumin concentrations showed middle correlation (r = 0.49) in normoalbuminemic dogs and high degree of correlation (r = 0.67) in hypoalbuminemic dogs. These results showed the importance of recognizing serum glucose concentration as well as total serum protein and albumin concentrations in the assay of canine serum fructosamine concentration.     

Pyrogallol poisoning of pigeons caused by acorns

Green acorns are known to contain high concentrations of pyrogallol. Here, we describe an extended case report of two pigeons found dead with a filled muscular stomach of acorns. The following pathologic findings were observed: irritation of mucosal membranes in the gastrointestinal tract, blackish discolored chyme, hyperemic organs, and general edemas. The muscular stomach (ventriculus) was filled with pieces of acorns, and the abdominal cavity contained bloody aqueous fluid. In order to uncover the cause of death, we determined pyrogallol in liver and kidney of one dead pigeon and in ventriculus contents of both pigeons by gas chromatography/mass spectrometry. A further aim of our study was to compare pathologic findings and pyrogallol concentrations in kidney, liver, and ventriculus of poisoned pigeons with those of healthy pigeons. The pyrogallol concentrations in samples of dead pigeons were 16-1200-fold higher than in control animals fed grass and maize-corn. Altogether, the acorn-filled ventriculus, the pathologic findings, the well nourished state, and the high pyrogallol concentrations in the dead pigeons suggest an acute pyrogallol poisoning by acorn. With respect to controls, we conclude that pyrogallol concentrations of 6 ng/g of kidney, 8 ng/g of liver, and 2 ng/g of gastric content do not affect the health of pigeons.