Glycosylated hemoglobin measurement in dogs and cats: implications for its utility in diabetic monitoring

The measurement of glycosylated hemoglobin (HbA₁) levels in humans is used to indicate the degree of long-term diabetic control. Using a commercially available kit for human HbA₁, values were obtained for normal and diabetic dogs and cats. The normal range established in dogs was broad and overlapped considerably with the range in diabetics. Under the assay conditions and with a limited number of diabetic animals, the test was not found to be of value for dogs or cats.     

Glycosylated hemoglobin in dogs: precision, stability, and diagnostic utility

The precision and stability of the ion exchange chromatography assay for canine glycosylated hemoglobin (HbA(1)) were examined. The coefficient of variation (CV) of within-run replicate assays was 1.3 to 2.6%; the CV of between-run duplicate assays was 3.1%. The mean HbA(1) content in 44 healthy dogs was 7.1% (SD = 1.1%, range = 5.1-9.7%). Paired aliquots of 12 blood samples were stored at 4 degrees C and 25 degrees C, and HbA(1) was measured on the day of collection and at 3, 5, and 7 days after collection. In the blood stored at 4 degrees C, no significant increase in the HbA(1) content was seen. No significant increase in HbA(1) content was found in the blood stored at 25 degrees C after 3 days, but dramatic increases were observed after 5 and 7 days of storage. No significant difference was observed in the HbA1 content in heart blood collected 18 hours after death from 9 dogs kept at 25 degrees C. The HbA(1) content was measured in 10 hospitalized diabetic dogs. Five of the dogs had received no insulin and all 5 had elevated HbA(1) values. The other 5 dogs had received insulin for 1 to 9 months; 2 of the 5 had increased HbA(1) content. The HbA(1) content was determined periodically for 9 months in one diabetic dog and it declined from 14% to 8.2%.     

IgM paraprotein interference with hemoglobin measurement using the CELL-DYN 3500

A 10-year-old male Labrador Retriever was presented for a 6-week history of polyuria, polydipsia, rear limb weakness, and ocular discharge. The dog had marked hyperproteinemia with an IgM monoclonal gammopathy, as determined by serum protein electrophoresis and immunoelectrophoresis. Cytologic findings in a lymph node aspirate were suggestive for lymphoma, which was confirmed and identified as B cell lineage by immunophenotyping and PCR antigen receptor rearrangement. In the CBC results, marked discrepancy was observed in the hemoglobin (HGB) concentration and MCHC obtained on a CELL-DYN 3500 analyzer (HGB 13.3 g/dL, MCHC 61.4 g/dL) as compared with an IDEXX LaserCyte analyzer (HGB 7.0 g/dL, MCHC 39.2 g/dL). To investigate this discrepancy, plasma was removed from an EDTA-anticoagulated blood sample from the patient, replaced with an equal volume of CELL-DYN diluent, and analyzed on the CELL-DYN. The resulting HGB (6.72 g/dL) and MCHC (33.5 g/dL) results were similar to those obtained initially on the LaserCyte. We concluded that precipitation of IgM paraprotein by the CELL-DYN lyzing reagent, which contains quaternary ammonium salts, falsely increased the spectrophotometric measurement of HGB on the CELL-DYN. The high MCHC was attributed to the false increase in HGB concentration. No interference with HGB measurement occurred with the LaserCyte, which uses a hypotonic solution to lyse RBCs before HGB determination. Paraprotein interference should be considered in veterinary patients with monoclonal gammopathy and unexpectedly high HGB and MCHC values.     

Evaluation of an automated system for hemoglobin measurement in animals.

In veterinary medicine, PCV determined by centrifugation of blood in a microhematocrit tube is the most common clinical test used to initially assess and monitor anemic and polycythemic animals. In contrast, blood hemoglobin (Hb) concentration, rather than PCV, is generally determined in human patients. One automated system photometrically measures blood Hb concentration after conversion of Hb to azide methemoglobin without dilution and was found to be a simple and accurate instrument for use in human medicine. We evaluated the system for its accuracy in measuring blood Hb concentration in animals by comparing it with standard techniques and for its suitability in veterinary practice. Blood samples, anticoagulated with potassium EDTA, from 78 healthy animals (33 dogs, 17 cats, 13 horses, and 15 cows) and 58 dogs and 4 cats with various blood abnormalities (10 anemia, 11 polycythemia, 21 lipemia, 16 leukocytosis, and 6 icterus) were analyzed. In all species, blood Hb concentration of healthy animals determined by the system was comparable to that measured by standard cyanmethemoglobin methods (ie, an automated counter; rI = 0.987 to 0.998 and a hemoglobin kit, rI = 0.946 to 0.993). The aforementioned system also yielded similar values to those obtained by use of standard methods in anemic, polycythemic, and icteric dogs and cats. Moreover, the system reads the absorbance at 2 wavelengths to correct for turbidity, and therefore, accurately measured Hb concentration in blood samples with severe lipemia (triglycerides concentration > 500 mg/dl) and marked leukocytosis (> 50,000 WBC/microliter), whereas other standard Hb techniques are known to give falsely high results.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of a commercial kit for measurement of glycosylated hemoglobin in canine blood

Total glycosylated hemoglobin (HbA(1)) was measured in canine blood samples by conventional macrocolumn ion exchange chromatography and with a commercial glycosylated hemoglobin kit. The two methods correlated well (r= 0.94, p < 0.001) and the reproducibility of the kit method was good. The commercial kit method is recommended for measurement of HbA(1) in canine blood.     

Glycosylated haemoglobin assay of canine blood samples

A method for assaying canine glycosylated haemoglobin was evaluated. The method is a turbidimetric inhibition immunoassay and the final reaction is bichromatically measured using a multichannel automatic analyser. Within-run coefficients of variation (2.07 to 4.46 per cent) were permissible, but between-run coefficients of variation (2.10 to 8.25 per cent) were slightly more elevated. The detection limit of this assay is 0.052 per cent. A sample dilution of 10 microliters of sample and 500 microliters of haemolysing reagent is recommended for routine analysis of canine blood samples. A normal reference interval of 1.39 +/- 0.70 per cent was obtained from the glycosylated haemoglobin analysis in 82 healthy dogs and no statistically significant differences in relation to age or gender were observed. Some changes in glycosylated haemoglobin concentrations were noted throughout the ovarian cycle, although differences between dogs were evident. Since this assay specifically measures the glycosylated haemoglobin content in canine blood samples, it could be very useful for monitoring diabetic dogs.     

Evaluation of three methods for measurement of hemoglobin and calculated hemoglobin parameters with the ADVIA 2120 and ADVIA 120 in dogs, cats, and horses

BACKGROUND: Besides flow cytometric detection of cellular hemoglobin (HGB) concentration, the ADVIA 2120 uses a novel cyanide-free colorimetric method to determine extracellular total HGB concentration. In human samples, the results are equivalent to those of the cyanmethemoglobin method on the ADVIA 120. Cyanide-free HGB measurement has not been evaluated in animal samples. OBJECTIVES: The aim of this prospective study was to compare the 3 methods of HGB analysis on the ADVIA 2120 and ADVIA 120 in blood samples from dogs, cats, and horses. METHODS: Consecutive fresh K(3)EDTA blood samples from 119 dogs, 113 cats, and 151 horses submitted to the Central Laboratory, Department of Veterinary Clinical Sciences, Justus-Liebig University Giessen, were included. A CBC was performed on each sample using the ADVIA 2120 and ADVIA 120. Colorimetric and cellular HGB concentrations and all calculated variables based on HGB measurement were compared using linear regression, Passing Bablok regression, and Bland Altman plots, using the ADVIA 120 as the reference method. RESULTS: In samples from all species, an excellent correlation was found for colorimetric HGB results (r=0.99). HGB measured with the cyanide-free method was overestimated on the ADVIA 2120 compared with the cyanide-based method on the ADVIA 120, with a mean proportional bias of -21.0% (dog), -22.0% (cat), and -19.4% (horse). The correlation of cellular HGB concentration between analyzers was excellent (r=0.99); however, imprecision was higher than for colorimetric methods. Excellent to fair agreement was found for all calculated variables. CONCLUSION: The cyanide-free method of HGB determination is appropriate for use in blood samples from animals, provided the proportional bias is considered.     

Glycosylated albumin and serum protein in diabetic dogs

Glycosylated albumin and glycosylated protein in serum were measured in 4 well-controlled diabetic dogs, 4 poorly controlled diabetic dogs, and 21 nondiabetic dogs. Concentrations of both glycosylated components in the well-controlled dogs were similar to those in nondiabetic dogs. Serum concentrations of glycosylated albumin and protein in the poorly controlled diabetic dogs were higher (P less than 0.001) than those of the nondiabetic and well-controlled diabetic dogs. Because of the essentially irreversible nature of the glycosylation reaction and the relatively short turnover time of albumin and other serum proteins, measurements of glycosylated serum components may provide an index of glycemia during the preceding days or weeks.     

Evaluation of three methods for measurement of hemoglobin and calculated hemoglobin variables with the ADVIA 120(R) and ADVIA 2120(R) systems in goats

The present study investigated 3 methods of hemoglobin (Hb) determination in goats using the ADVIA 120 and ADVIA 2120 systems. Ethylenediaminetetraacetic acid anticoagulated caprine blood samples (n = 40 goats) were subjected to Hb determination via the cyanmethemoglobin methods in both instruments and a novel, cyanide-free, colorimetric method with the ADVIA 2120. Statistical analysis of the data included a linear regression, Passing-Bablok regression, and Bland-Altman diagram. Colorimetric Hb results determined with both analyzers had excellent correlation (r = 0.98); however, a mean proportional bias of -19.1% was present in comparison to the reference method. There also was excellent agreement between cellular Hb concentrations when measured with both analyzers (r = 0.96), and the constant bias was close to zero. However, imprecision was higher compared to colorimetric methods. Excellent to fair agreement was evident for all calculated erythrocyte and Hb variables. Because of the excellent correlation between the ADVIA 120 and ADVIA 2120, the cyanide-free method of Hb determination could be used with caprine blood specimens; however, the proportional bias must be considered.     

Glycosylated haemoglobin in dogs: study of critical difference value

Measurement of glycated proteins can be of use in diagnosis and monitoring of diabetic dogs. Its use in monitoring can be facilitated by comparison of results with a reference interval derived from levels in normal dogs. In this study, a commercial immunoturbidometric assay was used to measure glycosylated haemoglobin in 15 normal dogs over a 5-week period. Following statistical analysis of the results a critical difference value of 0.38 per cent was obtained.     

The HemoCue® for point-of-care hemoglobin measurement and packed cell volume estimation in cats

Objective: To determine conversion formulas so that values from the HemoCue^® hemoglobinometer (Hb_HQ) could be equated to hemoglobin concentrations measured by the gold‐standard cyanomethemoglobin (Hb_CY) method and used for estimation of packed cell volume (PCV) in cats. Study design: Prospective, in vitro. Animals: Twelve healthy, adult, client‐owned cats. Interventions: The PCV of 12 parent blood samples was manipulated between ～3 and ～80% by removing or adding autologous plasma. Hemoglobin was measured by the Hb_CY method at a university clinical pathology laboratory and by the azidemethemoglobin method in a Hb_HQ. PCV was determined by micro‐centrifugation. Measurements and main results: Hemoglobin varied from 1–26 g/dL. Repeated‐measures regression of Hb_CY on Hb_HQ demonstrated that the Y‐intercept was not different from zero. When the regression was repeated, forcing the line through the origin the coefficient for converting Hb_HQ to Hb_CY was not significantly different from 1.0 (adjusted R²=99.7%). The conversion formula was Hb_CY=Hb_HQ. Using similar methods, the conversion formula for estimating PCV was PCV=3.1 Hb_HQ (adjusted R²=99.8%). Conclusion: Hemoglobin concentration measured by the HemoCue^® was indistinguishable from the (Hb_CY) method in feline blood over a wide range of hemoglobin concentrations. This study demonstrates that in cats PCV=3.1Hb_HQ. Clinical relevance: The HemoCue^® is useful for point‐of‐care hemoglobinometry and for estimating PCV in cats.     

Glycosylated Hemoglobin Concentration for Assessment of Glycemic Control in Diabetic Cats

Blood glycosylated hemoglobin (GHb) concentration was quantified in 84 healthy cats, 9 cats with stress-induced hyperglycemia, 37 cats with newly diagnosed diabetes mellitus, and 122 diabetic cats treated with insulin or glipizide. Diabetic control was classified as good or poor in insulin-treated or glipizide-treated cats based on review of history, physical examination findings, changes in body weight, and measurement of blood glucose concentrations. Blood GHb concentration was determined using an affinity chromatography assay. Mean blood GHb concentration was similar for healthy normoglycemic cats and cats with transient, stress-induced hyperglycemia, but was significantly (P < .001) higher in untreated diabetic cats when compared with healthy normoglycemic cats. Mean blood GHb concentration was significantly (P < .001) higher in 84 cats with poorly controlled diabetes mellitus when compared with 38 cats in which the disease was well controlled. Mean blood GHb concentration decreased significantly (P < .01) in 6 cats with untreated diabetes mellitus after insulin and dietary treatment. A similar significant (P < .01) decrease in mean blood GHb concentration occurred in 7 cats with poorly controlled diabetes mellitus after diabetic control was improved by an increase in insulin dosage from 1.1 ± 0.9 to 1.4 ± 0.6 U/kg/ 24 h and by feeding a diet containing increased fiber content and in 6 cats with transient diabetes mellitus 8.2 ± 0.6 weeks after discontinuing insulin treatment. There was a significant (P< .01) stress-induced increase in mean fasting blood glucose concentration and mean blood glucose concentration for 12 hours after administration of insulin or glipizide but no change in mean blood GHb concentration in 5 docile diabetic cats 12.2 ± 0.4 weeks after the cats became fractious as a result of frequent hospitalizations and blood samplings. Results of this study suggest that evaluation of blood GHb concentration may be a clinically useful tool for monitoring glycemic control of diabetes in cats.     

Beta-globin chain hemoglobin polymorphism and hemoglobin stability in black rhinoceroses (Diceros bicornis)

To evaluate the syndrome of acute intravascular hemolytic anemia in black rhinoceroses (Diceros bicornis), the hemoglobin of this species was evaluated by use of isopropanol- and heat-stability tests and was further characterized by electrophoretic studies. Samples were obtained from 22 apparently healthy captive North American black rhinoceroses, though 3 of the study animals had survived previous hemolytic events, and 3 others were parents of 3 offspring that had suffered hemolysis. The eastern African (Diceros bicornis michaeli) and the southern African subspecies (D b minor) were represented. Comparative samples were also obtained from 2 white (Ceratotherium simum) and 1 Indian (Rhinoceros unicornis) rhinoceroses. The hemoglobin of all 3 species appeared stable when tested by use of the heat and isopropanol methods. Thus, an unstable hemoglobin does not appear to be involved in the hemolytic crises of captive black rhinoceroses. Black rhinoceros hemoglobin had a striking polymorphism. Thirteen of the samples from black rhinoceroses had a single hemoglobin band, based on results of alkaline electrophoresis. Nine had, in addition to this major band, a slow (more cathodic) minor band that comprised about 10% of the total hemoglobin. Further studies indicated that the major band and the slower minor band may contain globin chains analogous to human beta- and delta-chains respectively; these bands have been tentatively designated B and C. Phenotypes B and BC are common, in a ratio of 4:3. A genetic mechanism is proposed that assumes beta b and beta c gene loci and that beta c-locus-expressed (beta c+) and beta c-locus-inhibited (beta c degrees) are common alleles for the beta c-locus.(ABSTRACT TRUNCATED AT 250 WORDS)     

血红蛋白粉对仔猪血液中血红蛋白、铁及转铁蛋白含量的影响

选择35日龄、体重相近的杜×长×大三元杂交仔猪84头,按遗传基础相似、公母各半的原则,随机分成4组,第I组为对照组,其余为试验组,每个处理组设3个重复,分别用0%、1%、2%、3%的血红蛋白粉替代其日粮中相应比例的鱼粉(其中试验Ⅲ组中替代2%的鱼粉和1%的豆粕),并进行了为期30d的饲养试验。试验结果表明,仔猪的日增重试验Ⅱ组和试验Ⅲ组显著高于试验Ⅰ组和对照组(P<0.05),且试验Ⅲ组显著高于试验Ⅱ组(P<0.05);仔猪的血红蛋白浓度随血红蛋白粉添加量的增加而成正相关;仔猪血浆中转铁蛋白的含量随日粮中血红蛋白粉添加比例的增加而上升,并且各组间差异是显著的(P<0.05);仔猪的血浆铁含量试验Ⅲ组显著高于其它组(P<0.05)。     

Detection of Glycosylated Proteins in Rabbit Oviductal Isthmus and Uterine Endometrium During Early Embryo Development

In this study, lectin histochemistry was performed on paraffin sections to compare carbohydrate expression of oviductal isthmus and uterine endometrium in rabbits during early embryo development. Rabbit embryos are surrounded not only by the zona pellucida but also by tubal secretion‐derived mucinous coat material, the mucin coat. Twenty sexually mature females were euthanized at 0 (pre‐ovulatory group) and 24, 72 and 96 h after insemination (pseudopregnancy group). The following lectin‐binding agents were used: Arachis hypogaea, Peanut (PNA) to label galactosyl (β‐1,3)N‐ acetyl‐galactosamine, Dolichos biflorus Agglutinin (DBA) to label galactosyl (β‐1,4)N‐ acetyl‐galactosamine, Lens curinaris (LCA) to label α‐‐mannose, α‐d ‐glucose and Pisum sativum agglutinin (PSA) to label α‐d ‐mannose, α‐d ‐glucose. Blood was collected by cardiac puncture, and direct enzyme immunoassay technique was used to measure progesterone concentration. A significant increase in total plasma progesterone concentrations was detected at 96 h post‐ovulation when compared with 0, 24 and 72 h post‐ovulation (2.9 ± 0.5 vs 0.5 ± 0.15, 1.6 ± 0.5 and 1.5 ± 0.4 ng/ml, at 96 h vs 0, 24 and 72 h post‐ovulation, respectively). No differences between pre‐ovulatory and pseudopregnant females were observed for glycoprotein localization in isthmus. In contrast, in the endometrium, differences in the glycoprotein detection between pre‐ovulatory and pseudopregnant stages were detected. PNA to label galactosyl (β‐1,3)N‐ acetyl‐galactosamine was not detected at the pre‐ovulatory stage, but its presence was detected at 24 h after ovulation. Both PSA and LCA to label α‐d ‐mannose, α‐d ‐glucose were only detected at 72 h after ovulation. DBA detection was similar for all stages of the reproductive cycle. Therefore, N‐acetyl‐galactosamine secreted from isthmus could be involved in the formation of the embryonic mucin coat. d ‐galactose (PNA), d ‐glucose and d ‐mannose (PSA and LCA) might be crucial for the implantation period.