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Plasma protein electrophoresis in birds: comparison of a semiautomated agarose gel system with an automated capillary system.

Abstract: Plasma agarose gel electrophoresis (AGE) is recognized as a very reliable diagnostic tool in avian medicine. Within the last 10 years, new electrophoresis techniques such as capillary zone electrophoresis (CZE) have emerged in human laboratory medicine but have never been investigated in birds. To investigate the use of CZE in birds and to compare it with AGE, plasma samples from 30 roosters (Gallus gallus), 20 black kites (Milvus migrans), and 10 racing pigeons (Columba livia) were analyzed by both AGE and CZE. For the 3 species studied, values determined by AGE and CZE were well correlated for albumin and beta and gamma fractions whereas other values differed significantly. Values for alpha-3 fraction in the rooster, alpha-1 fraction in the black kite, and alpha fractions in the pigeon obtained by AGE were very well correlated with the prealbumin fraction values obtained by CZE. Repeatability and reproducibility appeared higher with CZE than with AGE. Although the interpretation of CZE electrophoresis patterns seems to produce results similar to those obtained with AGE, some proteins present in the alpha fraction measured with AGE migrated to the prealbumin fraction found with CZE. Although CZE requires the use of specific reference intervals and a much higher sample volume, this method has many advantages when compared with AGE, including better repeatability and reproducibility and higher analysis output.

Key words: agarose gel electrophoresis, capillary zone electrophoresis, plasma proteins, protein electrophoresis, avian, birds

Introduction

In human medicine, serum protein electrophoresis techniques have been used for 5 decades. (1-3) Although the use of protein electrophoresis in avian medicine is far more recent and is still in its infancy, publications made during the last 15 years show that it is a very reliable diagnostic test in birds. (4-6) Indeed, plasma proteins have many functions in health and disease. In the case of an inflammatory condition, acute phase protein plasma levels increase and are of clinical interest in the diagnosis of various diseases in birds. (7,8) Within the last 10 years, new electrophoresis techniques, such as capillary zone electrophoresis (CZE) have emerged in the field of human medicine. (2,9) Such advances should also be investigated in birds.

Since Tiselius's pioneering work, (10) practices in human laboratory clinical medicine have always closely followed advances in protein electrophoresis. Nowadays, agarose gel electrophoresis (AGE) is the most currently used technique. (3,11) In 1997, Sebia developed a semiautomated electrophoresis system called Hydrasys (Sebia, Norcross, GA, USA). This system further improved the reproducibility of AGE by automating most steps of the procedure, including sample application, migration, and staining. (12) However, despite such semiautomated systems, AGE remains quite labor-intensive, resulting in limited analytical performance and throughput. (13)

Over the last decade, CZE has emerged as a powerful diagnostic tool in the field of human diagnostics and represents a major advance in electrophoresis technology for clinical applications. (9,13-15) This technique, first introduced by Hjerten in 1967, consists of the separation of charged molecules in small capillary tubes. Because heat can readily radiate from the tubes, extremely high voltages can be used without overheating the samples. (16) The use of such high voltages shortens the analysis times. (17) Protein analysis is performed in a free buffered solution, and with this system, the electro-osmotic velocity exceeds the electrophoretic mobility of the proteins, which therefore migrate toward the cathode instead of the anode. Real-time detection and quantification of protein fractions is based on the direct detection of peptide bonds by way of ultraviolet (UV) light absorption through the capillary wall. (9,13-15,17)

The first automated capillary electrophoresis system, the Paragon CZE2000 (Beckman Coulter Inc, Fullerton, CA, USA) was commercialized in 1994. In 2001, Sebia developed its own CZE system, Capillarys. Such systems are particularly helpful in clinical laboratories that have to deal with a large daily workload of serum protein electrophoresis (up to 100/h with Sebia Capillarys2. (12) These systems are indeed fully automated and require only minimal human intervention. In human medicine, various publications have dealt with the topic of CZE and compared its technique with that of AGE. These studies show good correlations between the results obtained with both techniques, with the exception of alpha-1 and beta fractions. The reproducibility of CZE appears to be superior to that of AGE. (2,9,11,13,15) Capillary zone electrophoresis is at least equivalent to AGE, and the interpretation of its electrophoregrams is similar to that for AGE. (15) In dogs and cats, however, visual appearance of CZE profiles and quantitative information provided by this technique differ from those of AGE. (18)

In birds, various publications have documented the use of paper electrophoresis, cellulose acetate electrophoresis, or AGE for research purposes or as diagnostic tools. (19-30) Comparative studies of these techniques have also been made. (19) However, our review of the literature has failed to reveal any study in which the use of CZE has been studied for birds.

The objective of this study was therefore to describe the use of Sebia Capillarys2 CZE for birds and to compare its results with those obtained using Sebia Phoresis AGE. The study was also designed to produce the first CZE reference intervals for birds and to measure the reproducibility of this technique.

Material and Methods

Experimental animals

Plasma samples from 30 roosters (Gallus gallus), 20 black kites (Milvus migrans), and 10 pigeons (Columba livia) were analyzed by using the Sebia AGE and CZE systems. The study was conducted on 30 SY33 line roosters held at the Institut National de Recherche Agronomique Tours-Nouzilly research station (France). These birds were 3 years old and were housed in individual cages. To broaden the scope of our investigation of capillary electrophoresis in birds, 20 black kites held at the Academie de Fauconnerie du Puy du fou (France), and 10 pigeons held at the zoological park of Cleres (France), also were included in the study. Both the pigeons and the black kites were housed in aviaries. All blood samples were obtained on the occasion of veterinary screening protocols. All of these birds were examined and determined to be clinically healthy.

Samples

Blood samples were taken from the right jugular vein of the roosters and black kites and from the brachial vein of the pigeons by using 23-gauge needles and 5-mL syringes. Samples of 2 mL of blood were drawn from each bird, collected into lithium heparin tubes (Venosafe evacuated blood collection tubes, Terumo, Leuven, Belgium), and centrifuged at 3000g for 5 minutes. Analyses were done on the resulting plasma samples because plasma usually is considered preferable to serum in birds and plasma is less prone to fibrin clots and hemolysis than serum and contains fibrinogen, a protein characteristic of the acute phase of inflammatory conditions. (4,31) Samples did not show any hemolysis or lipemia. Two aliquots of each plasma sample were then stored in cryotubes (-20[degrees]C [-4[degrees]F]) until they were analyzed (Micronic Systems, Lelystad, Holland). Samples were then thawed and rehomogenized by gentle mixing, 1 hour before analysis. All blood samples were collected on the same day and were therefore exposed to similar freezing conditions.

Measurement of total protein concentration

Total protein concentration was determined by the Biuret reaction, using a Roche Integra 400 wet chemistry analyzer (Roche Diagnostics GmbH, Mannheim, Germany). Readings were made at a wavelength of 552 nm.

Agarose gel electrophoresis

Agarose gel electrophoresis of plasma proteins was done by using a Hydrasys semiautomated system (Sebia, Evry, France), using the Hydragel protein 15/30 set (Sebia). It was operated according to the manufacturer's instructions, using version 7.00 F0.1. of the system software. Sample aliquots of 10 [micro]L of plasma were manually distributed onto the sample template and allowed to diffuse for a period of 5 minutes in a wet chamber. Application of the samples to the gel, electrophoresis, and drying of the gel were all performed automatically in the migration compartment of the instrument. The temperature was maintained at 20[degrees]C (68[degrees]F) by using a Peltier device during the complete migration process, and drying was obtained by heating the gels to 65[degrees]C (149[degrees]F). Electrophoretic separation was obtained on 8 g/L agarose gels in a Tris-barbital buffer, at pH 9.2 and a constant power level of 20W, until 33 V.h had been accumulated. Once dried, the gels were manually transferred to the staining compartment of the instrument where amido-black staining, destaining, and drying were performed automatically. Once these operations had been completed, the gels were scanned with a high-resolution Epson perfect V700 photo scanner (Epson France, Nanterre, France). Electrophoretic curves and dosages of the different fractions were acquired using Phoresis software, version 5.50 (Sebia). Protein fractions were determined by referring to other publications. (25,28,30)

By convention, the albumin : globulin ratio (A/G) in birds is calculated by dividing the sum of the prealbumin and albumin fractions by the sum of the globulin fractions. (4,31)

Capillary zone electrophoresis

Capillary zone electrophoresis of plasma proteins was carried out on a Capillarys2 automated system (Sebia). This system was operated according to the manufacturer's instructions under version 6.00 of the supplied software.

Sample tubes were manually installed into racks (up to 13 racks of 8 sample tubes). A minimum volume of 140 [micro]L was used. All of the following steps were performed automatically. The samples were diluted to a 1:5 ratio with the migration buffer in dilution segments (40 [micro]L of plasma to a final volume of 200 [micro]L). The samples were then hydrodynamically injected using an anodic depression of 80 millibars for 4 seconds (<1 nL, representing less than 1% of the total volume of the capillary tube). With the Capillarys protein 6 reagent set (Sebia), electrophoretic separation of the protein fractions was obtained by applying a 7.5-kV voltage for about 4 minutes to 8 fused-silica capillaries (17.5 cm in length, 15.5 cm in effective length; 25 [micro]m internal diameter) in a pH 10 buffer. The temperature was maintained at 35.5[degrees]C (95.9[degrees]F) by using a Peltier controller.

Real-time detection and quantification of protein fractions was based on the direct detection of peptide bonds, resulting from UV light absorption through the capillary wall. Detection was performed at 200 nm, in an optical cell placed at the cathodic end of the capillary tube and connected to the detector by means of 8 optical fibers. Electrophoretic curves and dosages of the different fractions were acquired using version 6.00 of the Phoresis software (Sebia).

Repeatability and reproducibility

One rooster sample was chosen at random and was used to investigate the repeatability and reproducibility of AGE and CZE.

For AGE, repeatability within a run was estimated by interpreting the plasma patterns of 30 aliquots of the same sample run on the same gel. Interrun reproducibility was estimated by interpreting the plasma patterns of 8 aliquots of the same sample run on separated gels.

For CZE, repeatability within a run was estimated by interpreting the plasma patterns of 8 aliquots of the same sample run on the same rack. Interrun reproducibility was estimated by interpreting the plasma patterns of 6 aliquots of the same sample run on different racks.

The coefficients of variation (CVs) were calculated for each fraction.

Statistical analysis

Medcalc 12.1.4.0 software (Medcalc software, Mariakerke, Belgium) was used for all analyses. The normality of the data from the 3 studied species was tested with the Kolmogorov-Smirnov procedure. For each method and electrophoretic parameter, descriptive statistics (median [minimum-maximum]) were used to illustrate data distribution for the whole set of samples. In view of the small size of the studied populations, Wilcoxon's signed rank was used to compare the 2 different electrophoresis techniques, taking into account that such a test is not applicable if a proportional error is present. (32) As the data were found to be normally distributed, Pearson's correlation matrix was used to assess correlations between results for both methods in all of the studied species. As in Cray et al, (33) Pearson's correlation coefficients (r) were considered to be excellent (0.9-1.0), high (0.70-0.89), good (0.50-0.69), low (0.30-0.49), or poor (<0.29).

The study of AGE and CZE accuracy was made by calculating the CVs. Other authors have indeed stated that CV analysis is the recommended approach for assessing repeatability and reproducibility of chemistry tests. (6)

Agreement between CZE and AGE was evaluated by Passing-Bablok regression analysis and Bland-Altman difference plot. Passing-Bablok analysis was used to point out the presence of constant and/or proportional errors. When the 95% confidence interval (CI) included intercept and slope values of 0 and 1, respectively, agreement was considered to be good. If one or both of the 95% CIs did not include the 0 and 1 values, a constant (intercept different from 0) and/or proportional (slope different from 1) error was present, and agreement was considered to be poor. (18,32) Bland-Altman analysis was used to calculate the mean difference between AGE and CZE for each fraction in the 3 species and to judge the acceptability based on inherent imprecision of both methods in roosters. The intrarun and interrun CVs were combined to calculate the total CV for each fraction in this species. (32,33) If more than 95% of the differences between the 2 techniques were within the expected combined imprecision (0 + 1.96, CV; 0 - 1.96, CV), results were not considered to be different. (32,33)

Results

Protein electrophoresis patterns

The AGE patterns of the rooster were divided into 8 fractions, which were then classified into 1 prealbumin fraction, 1 albumin fraction, 3 alpha fractions, 2 beta fractions, and 1 gamma fraction (Fig 1, Table 1). The electrophoresis patterns obtained with CZE were found to be quite similar to those obtained with AGE, and fractions were defined the same way (Fig 1, Table 1). The study of the correlations between values obtained, using both CZE and AGE, revealed good to excellent correlations for the A/G ratio, albumin, and alpha-1, alpha-3, beta-1, beta-2, and gamma fractions (Table 1). However, the alpha-3 values obtained with AGE were well correlated with the prealbumin values obtained with CZE (n = 30, r = .519, P < .01). With the exception of the beta-2 fraction, the values obtained with AGE and CZE for the remaining fractions were found to be significantly different (Table 1).

Analysis of the reference intervals of intercepts and slopes determined by the Passing-Bablok regression analysis identified proportional errors for alpha-l, gamma, and A/G values and a constant error for alpha-3 values (Table 1). Analysis of the data obtained by the Bland-Altman difference plot in the light of combined CVs pointed out the methods could not be considered identical within inherent imprecision of both methods (Table 1).

The AGE and CZE patterns of the black kites were divided into 6 fractions (Fig 1, Table 1). These were classified into 1 prealbumin fraction, 1 albumin fraction, 2 alpha fractions, 1 beta fraction, and 1 gamma fraction. The albumin fraction was identified as having the strongest anodal peak. However, with AGE, the electrophoresis patterns of the black kites were characterized by the presence of a strong alpha-I peak, similar in intensity to the albumin peak. Such a strong peak was not observed in CZE patterns (Fig 1, Table 1). The study of the correlations between values obtained using both CZE and AGE revealed good to excellent correlations for the A/G ratio, albumin, and alpha-l, alpha-2, beta, and gamma fractions (Table 1). Furthermore, the alpha-1 values obtained with AGE were highly correlated with the prealbumin values obtained using CZE (n = 30, r = .848, P < .001). The values obtained with AGE and CZE were all found to be significantly different (Table 1).

Analysis of the reference intervals of the intercepts and slopes determined by the Passing-Bablok regression analysis identified proportional errors for gamma and A/G values and constant errors for alpha-1 and gamma values (Table 1). Furthermore, no linear relation could be found between prealbumin values from both techniques. Analysis of the data obtained by the Bland-Altman difference plot pointed out large mean differences between AGE and CZE, especially for the prealbumin and alpha-1 values (Table 1).

The AGE patterns of the pigeons were divided into 6 fractions (Fig 1, Table 1). These were then classified into 1 prealbumin fraction, 1 albumin fraction, 1 alpha fraction, 2 beta fractions, and 1 gamma fraction. The albumin fraction was identified as having the strongest anodal peak. Similar to the black kites, with AGE the electrophoresis patterns of the pigeons were characterized by the presence of a second anodic peak at the alpha-1 position, comparable in intensity with the albumin peak (Fig 1).

The CZE electrophoresis patterns of the pigeons were divided into 9 fractions (Fig 1, Table 1). These electrophoresis patterns were then also found to present 2 intense anodic peaks. By comparing the height of the peaks obtained using AGE and CZE, we chose to classify the most anodic peak with the prealbumins, and the second peak with the albumin. The correlations confirmed this choice, since the resulting albumin fraction was strongly correlated with that determined by AGE (n = 10, r = .99, P < .001). Furthermore, a strong correlation between the prealbumin fraction obtained by CZE and the alpha fraction obtained by AGE was stressed (n = 10, r = .98, P < .001). The beta and gamma fractions were named by comparison with the electrophoresis patterns obtained on agarose gel. The beta-1 and beta-2 fractions were found to be highly correlated with the beta-1 and beta-2 fractions obtained on agarose gel (Table 1). The sum of the 3 alpha fractions and the sum of the 2 gamma fractions obtained on CZE were highly correlated with the alpha and gamma fractions obtained by AGE, respectively (Table 1). The values obtained with AGE and CZE were all significantly different (Table 1).

Analysis of the reference intervals of the intercepts and slopes determined by the Passing-Bablok regression analysis identified a proportional error for beta-2 values (Table 1). Furthermore, no linear relation could be found between prealbumin, alpha, and A/G values from both techniques. Analysis of the data obtained by the Bland-Altman difference plot pointed out large mean differences between AGE and CZE, especially for the prealbumin and alpha values (Table 1).

Repeatability and reproducibility

The results of the intrarun repeatability and interrun reproducibility, obtained for both AGE and CZE, are given in Table 2. The CVs ranged from 1.8% to 9.9% for AGE, and 0.9% to 4.2% for CZE. The accuracy therefore appeared to be higher in the case of CZE than with AGE. With each technique, both repeatability and reproducibility were very good for strong and well-defined fractions. However, they were lower for poorly defined fractions, i.e., alpha-1, alpha-2, beta-2, and gamma fractions in AGE and alpha fractions in CZE. The repeatability and reproducibility achieved for the fractions of weak intensity were also generally weak, as was the case for example with the prealbumin and alpha-2 fractions obtained with both techniques.

Discussion

To our knowledge, this study is the first to have dealt with plasma protein CZE in birds. Our data, therefore, represent the first set of baseline information relative to the use of CZE in avian medicine. Potential weaknesses of this study are the small sample sizes (less than the 40 usually recommended), especially for black kites and pigeons, and the use of only normal samples. (32)

For the 3 studied species, generally we found a good degree of correlation between the results obtained from the 2 techniques, in particular for the albumin, beta, and gamma fractions. These correlations do not mean that these fractions are exactly the same but that some of the most abundant proteins constituting them are the same. The interpretation of CZE electrophoretic patterns may therefore be quite similar to that of AGE patterns. However, the results from correlations between fractions indicate that, for the 3 species studied, some of the alpha-fraction proteins in AGE could migrate to the prealbumin fraction in CZE. Indeed, we observed a good degree of correlation between the alpha-3 fraction values in AGE and those corresponding to the prealbumin fraction in CZE for the rooster, and an excellent degree of correlation between the alpha-1 fraction values and those corresponding to the prealbumin fraction in CZE for the black kites. This phenomenon is even more obvious in the pigeon with the alpha fraction. In a previous study based on pigeons and black kites, this alpha fraction has been demonstrated to be mainly composed of apolipoprotein A1, a negative acute-phase protein. (34) In this species, the strength of the resulting prealbumin peak obtained with CZE can lead to confusion. Such an intense anodic fraction could in effect be mistakenly identified as the fraction corresponding to albumin. A phenomenon of such an intensity has, as far as we know, never been documented in humans, as AGE and CZE patterns are considered to be very similar. (2,9,11,13,15) Indeed, for human laboratory medicine, buffered solutions have been specially designed so that CZE patterns have the same general aspect as AGE patterns. (17)

For some fractions, such as the alpha fractions, correlations obtained with AGE and CZE were weaker than for other fractions. Manual separation of such poorly defined fractions of such weak intensity is likely highly subjective for the technician. (6,35) As a consequence, for such fractions the resulting relative error margin is significant.

Passing-Bablok regression analysis and Bland-Altman difference plots showed significant discrepancies between the 2 techniques for prealbumin and alpha, beta, and gamma fraction values, depending on the species. The analysis of the acceptability based on inherent imprecision of both techniques in the rooster's data and the Wilcoxon's test results stress that the 2 methods cannot be considered to be identical, thus exposing the need for specific reference intervals for the correct interpretation of capillary electropherograms. As previously discussed, such differences could be related to differences in electrophoretic mobility between certain proteins, depending on which technique is used. They could also be related to differences in the affinity of certain proteins for the dyes used in AGE. Indeed, in human laboratory medicine, comparisons between CZE, immunonephelometry, and colorimetry suggest that direct quantification of albumin and alpha-1 globulin by UV absorption is more accurate than protein staining. (2,9) In the alpha-1 fraction, for instance, the high sialic acid content of the alpha-1 acid glycoprotein has been shown to interfere with the binding of dyes in gel-based methods, whereas UV absorption used in CZE is not affected by these sugar moieties. (36)

Because of the higher resolution of CZE compared with AGE, (2) some fractions were separated by CZE and not identified in AGE. The interpretation of such fractions is currently unknown and should be investigated in further studies.

By convention, the A/G ratio in birds is defined as the sum of the prealbumin and albumin fractions divided by the sum of the globulin fractions. (4,37) Taking into account the localization of proteins at the level of the prealbumin fraction in CZE that initially were present in the AGE alpha fraction, the A/G ratios defined for AGE cannot be compared with those obtained with CZE. It may therefore be more appropriate to define the A/G ratio in birds as that given by the albumin fraction divided by the sum of the globulin fractions, including the prealbumin in the globulins. (6)

The study reported here stresses that in roosters, the repeatability and reproducibility are higher with CZE than with AGE. This is consistent with publications concerning human laboratory medicine. (2,9,36) As reported by Bienvenu et al (2) in humans, the resolution achieved is higher and the clarity of the electrophoresis bands is better with CZE than with classic methods. The improved repeatability and reproducibility may furthermore be related to the complete automation of the analytical procedure and to the fact that in CZE, protein absorbance is measured directly by UV absorption instead of being determined indirectly from the amount of dye adsorbed onto the protein. (36) However, further studies should assess more thoroughly repeatability and reproducibility of CZE in pathologic avian samples.

For both techniques, the repeatability and reproducibility figures determined in this study are generally poorer than those given in publications related to human electrophoresis using the same equipment, (12,13,36) or in Sebia's user's manuals of the Protein 6 and Hydragel protein kits (Capillarys protein 6-2007/06; Hydragel 7, 15, and 30 protein-2005/5). This is probably related to the fact that the fractions are better separated from one another on the human electropherograms than on those obtained from birds, as reported in our study. The weak and poorly separated fractions are also those determined with the lowest accuracy. Manual separation of such fractions by the technician may indeed be more subjective, and the smaller the fraction is, the comparatively more significant the error becomes. (6,35) This may explain why, in contrast, the repeatability and reproducibility of strong and well-defined fractions such as albumin are excellent.

With regard to the poor accuracy with which the prealbumin fraction is determined using AGE, it was found that in certain cases, despite the care taken to clean the gels before curve acquisition, the electrophoresis curve did not completely return to the baseline, thus leading to overestimation of this fraction. This phenomenon was not noticed with CZE.

The analysis of electrophoresis patterns obtained from both techniques has finally enabled significant interspecies differences to be revealed. The electrophoresis patterns of the 3 species chosen for this study are in effect very different. Some authors have already discussed these significant interspecies differences for classic techniques, (4,27,33,38,39) and all practitioners should be aware that the electropherogram of an individual must always be interpreted by comparing it with a normal electrophoregram for the given species, or better still with personal reference intervals.

Further studies should be carried out to analyze plasma protein electrophoresis in other bird taxa with CZE, in particular for species regularly seen in veterinary practices or zoological gardens. Further studies should also investigate the effects of hemolysis and lipemia as interference factors and the effects of sample conservation on avian capillary electrophoregrams, as has been assessed in humans. (9)

Although the use of CZE requires establishing reference intervals for each species, it allows considerable advantages over traditional gel-based methods. Capillary zone electrophoresis doesn't require any matrix for protein separation. Apart from its improved repeatability and reproducibility, when compared with agarose gel or cellulose acetate methods, it can therefore offer considerable technological progress to the laboratories. Systems to run CZE such as Capillarys2 are indeed fully automated (bar-code identification of the samples, automation of all analytical steps) and thus require almost no technical manipulation. Such automated machines allow very high analysis rates to be used and are thus very well suited to laboratories that have high workloads. In our study, the Capillarys2 had an output of 80 samples/h when the samples were analyzed in a single batch, whereas with the Hydrasis system, processing of the first series took 45 minutes, and 25 minutes for the following series, with 30 plasma samples being simultaneously analyzed per batch.

Capillary electrophoresis nevertheless has drawbacks. It requires a larger sample size than AGE (14 times more in this study), which is a major disadvantage when dealing with small birds. However, because the normal packed cell volume of birds ranges between 35% and 55% and because blood samples representing 1% or less of a bird's body weight can usually be withdrawn from a healthy bird without detrimental effects, (40) performing CZE protein electrophoresis for a 30-g bird such as a budgerigar (Melopsittacus undulatus) may be possible, even though it should be considered as a limit. Furthermore, because it makes use of UV detection for direct protein quantification via peptide bonds, any substance in the plasma, such as a radio-opaque agent or sulfamethoxazole, which absorbs light at this wavelength, has the potential to produce an artifact. This is not the case with conventional gel-based methods, where quantification of the protein fractions is based on dye binding (amido-black in this study), which has a great affinity for proteins. (36,41,42) With birds, it would be highly interesting to verify whether the dietary supplementation of certain species held in captivity, such as flamingos (Phoenicopterus species) or the scarlet ibis (Eudocimus rubber), with synthetic carotenoids such as canthaxanthin, would produce artifacts with CZE.

Acknowledgments: We thank the Institut National de Recherche Agronomique in Tours-Nouzilly for the rooster blood samples they provided, Mr J. L. Liegeois from the Academie de Fauconnerie du Puy du Fou (France) for providing the black kite blood samples, Sebia for performing the CZE analyses, P. Trolliet from Sebia for his invaluable technical support, the team from Bio VSM, and K. Naudin from Bio VSM who carried out total protein measurements. We are grateful to the Museum National d'Histoire Naturelle and the Conseil General de Seine Maritime (France) for their financial support. We also appreciate the help provided by TechTrans in proofreading this document.

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Yannick Roman, DVM, PhD, Marie-Claude Bomsel-Demontoy, DVM, PhD, Julie Levrier, MSc, Daniel Chaste-Duvernoy, PharmD, and Michel Saint Jalme, PhD

From the Museum National d'Histoire Naturelle (MNHN), Departement des Jardins Botaniques et Zoologiques (DJBZ), Le Parc de Cleres, 32 avenue du Parc, 76690 Cleres, France (Roman, Levrier); MNHN, DJBZ, Menagerie du Jardin des Plantes, 57 rue Cuvier, 75005 Paris, France (Bomsel-Demontoy); Laboratoire Bio-VSM, 3 bis rue Pierre Mendes-France, 77200 Torcy, France (Chaste-Duvernoy): and MNHN, Departement Ecologie et Gestion de la Biodiversite, UMR 5173--Conservation des Especes, Restauration et Suivi des Populations--MNHN, Centre National de Recherche Scientifique, Paris IV, 57 rue Cuvier, 75005 Paris, France (Saint Jalme).

Table 1. Plasma protein electrophoresis fraction values and
summary of statistical analysis comparing AGE and CZE in
roosters, black kites, and racing pigeons. The AGE and CZE
results are expressed as median and ranges of the fraction
values. Wilcoxon's signed rank test results are represented
as asterisks in the CZE column.

 Values by species         AGE (g/dL)              CZE (g/dL)

Roosters (n = 30)
  Prealbumin            0.77 [0.26-1.90]      1.99 [1.29-2.801 ***
  Albumin              13.40 [10.52-16.81]   13.98 [10.92-18.02] ***
  Alpha-1               0.74 [0.51-1.78]      2.19 [1.62-3.33] ***
  Alpha-2               1.21 [0.87-1.86]      1.08 [0.84-1.50] *
  Alpha-3               6.31 [5.57-7.66]      3.51 [2.84-5.15] ***
  Beta-1                9.73 [6.56-14.68]     7.69 [4.61-11.54] ***
  Beta-2                2.80 [1.59-3.97]      2.55 [1.43-4.10] NS
  Gamma                 2.38 [1.65-4.48]      4.60 [2.29-8.02] ***
  A/G                   0.61 [0.40-0.80]      0.75 [0.47-0.98] ***
Black kites (n = 20)
  Prealbumin            0.49 [0.25-0.841      5.88 [3.60-8.66] ***
  Albumin              14.07 [10.78-18.43]   16.91 [13.14-21.97] ***
  Alpha-1              12.54 [9.16-15.91]     3.70 [2.46-5.02] ***
  Alpha-2               0.98 [0.80-1.38]      1.31 [1.01-1.73] ***
  Beta                  5.16 [4.09-6.31]      4.25 [3.02-5.38] ***
  Gamma                 5.09 [3.23-6.79]      5.89 [3.39-8.44] ***
  A/G                   0.62 [0.54-0.72]      1.59 [1.21-1.95] ***
Pigeons (n = 10)
  Prealbumin            0.73 [0.56-1.01]     11.22 [9.11-14.52] **
  Albumin               9.44 [8.08-12.89]     8.47 [7.30-11.77] **
  Alpha/Alpha
  1+2+3                10.81 [9.04-14.08]     2.94 [2.48-3.98] **
  Beta-1                3.33 [2.66-1.54]      2.52 [1.68-3.48] **
  Beta-2                1.56 [1.23-2.521      0.71 [0.52-1.42] **
  Gamma/
  Gamma 1+2             1.52 [0.67-1.83]      1.75 [0.83-2.47] **
  A/G                   0.61 [0.51-0.77]      2.49 [2.27-2.83] **

                       Spearman's
                       correlation   Passing-
 Values by species     coefficient    Bablok

Roosters (n = 30)
  Prealbumin               NS           NE
  Albumin               .967 ***        NE
  Alpha-1               .632 ***        PE
  Alpha-2                  NS           NE
  Alpha-3               .812 ***        CE
  Beta-1                .886 ***        NE
  Beta-2                .822 ***        NE
  Gamma                 .862 ***        PE
  A/G                   .952 ***      PE, CE
Black kites (n = 20)
  Prealbumin               NS           NL
  Albumin               .944 ***        NE
  Alpha-1               .803 ***        CE
  Alpha-2               .518 *          NE
  Beta                  .903 ***        NE
  Gamma                 .941 ***      CE, PE
  A/G                   .509 *          PE
Pigeons (n = 10)
  Prealbumin               NS           NL
  Albumin               .991 ***        NE
  Alpha/Alpha
  1+2+3                 .726 *          NL
  Beta-1                .945 ***        NE
  Beta-2                .851 **         PE
  Gamma/
  Gamma 1+2             .947 ***        NE
  A/G                      NS           NL

                        Bland-Altman        Bland-Altman
                            mean           % differences
                         difference      within imprecision
 Values by species        g/dL (%)             limits

Roosters (n = 30)
  Prealbumin            -1.19 (-88.3)          10.0
  Albumin               -0.65 (-4.6)           70.0
  Alpha-1               -1.40 (-95.4)           0.00
  Alpha-2                0.13 (10.1)           80.0
  Alpha-3                2.90 (59.2)            0.00
  Beta-1                 2.18 (25.6)            6.7
  Beta-2                 0.12 (4.1)            93.3
  Gamma                 -2.19 (-55.6)           0.00
  A/G                   -0.14 (-20.2)           0.00
Black kites (n = 20)
  Prealbumin            -5.70 (-165.8)
  Albumin               -2.93 (-18.7)
  Alpha-1                8.80 (109.3)
  Alpha-2               -0.32 (-26.7)
  Beta                   0.87 (18.8)
  Gamma                 -0.83 (-14.4)
  A/G                   -0.93 (-84.9)
Pigeons (n = 10)
  Prealbumin           -10.60 (-173.7)
  Albumin                1.14 (12.1)
  Alpha/Alpha
  1+2+3                  8.00 (113.5)
  Beta-1                 0.87 (29.4)
  Beta-2                 0.95 (74.8)
  Gamma/
  Gamma 1+2             -0.31 (-21.9)
  A/G                    -1.9 (-120.8)

Abbreviation: AGE indicates agarose gel electrophoresis; CZE,
capillary zone electrophoresis; NS, not significant; NE, no
error; PE, proportional error; CE, constant error; A/G, albumin:
globulin; NL, no linearity.

* P < .05; ** P < .01; *** P < .001

Table 2. Intrarun repeatability and interrun reproducibility of
AGE and CZE in roosters. Repeatability within a run  was
estimated by interpreting the plasma patterns of aliquots of the
same sample run on the same rack. Interrun  reproducibility was
estimated by interpreting the plasma patterns of aliquots of the
same sample run on different racks.

                                 AGE

                      Intrarun         Interrun
                   repeatability    reproducibility
Fraction           CV, % (n = 30)    CV, % (n = 8)

Prealbumin              9.1               8.3
Albumin                 1.8               2.2
Alpha-1-globulin        8.0               9.1
Alpha-2-globulin        9.9              11.1
Alpha-3-globulin        1.7               1.5
Beta-l-globulin         2.5               3.9
Beta-2-globulin         7.5               8.9
Gamma-globulin          6.5               6.3
A/G ratio               2.7               3.2

                                CZE

                     Intrarun         Interrun
                   repeatability   reproducibility
Fraction           CV, % (n = 8)    CV, % (n = 6)

Prealbumin              4.2              4.6
Albumin                 0.9              1.1
Alpha-1-globulin        2.6              2.8
Alpha-2-globulin        3.4              2.9
Alpha-3-globulin        3.3              4.5
Beta-l-globulin         0.8              1.5
Beta-2-globulin         1.7              4.1
Gamma-globulin          1.9              2.4
A/G ratio               1.9              2.1

Abbreviation: AGE indicates agarose gel electrophoresis; CZE,
capillary zone electrophoresis; CV, coefficient of variation;
A/G,  albumin: globulin.
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Title Annotation:Original Studies
Author:Roman, Yannick; Bomsel-Demontoy, Marie-Claude; Levrier, Julie; Chaste-Duvernoy, Daniel; Saint Jalme,
Publication:Journal of Avian Medicine and Surgery
Article Type:Report
Date:Jun 1, 2013
Words:6830
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