Research Article: Capillary hemoglobin electrophoresis of healthy and anemic dogs: Quantification, validation, and reference intervals of hemoglobin fractions

Date Published: September 4, 2019

Publisher: Public Library of Science

Author(s): Ioannis L. Oikonomidis, Theodora K. Tsouloufi, Mathios E. Mylonakis, Maria Kritsepi-Konstantinou, J Francis Borgio.


Despite the advances in canine medicine and the rapid gaining of attention of canine models in biomedical field and particularly in hemoglobin genes research, the studies on canine hemoglobin composition are sparse with ambiguous findings. Our aim was: i) to investigate the electrophoretic pattern of canine hemoglobin and the possible effects of age, sex, and anemia using a capillary electrophoresis assay, and ii) to validate this assay and calculate reference intervals (RIs) for canine hemoglobin fractions. Blood samples were collected from 53 healthy and 42 dogs with regenerative and non-regenerative anemias. The Sebia Capillarys 2 flex-piercing was used for hemoglobin analysis and it was validated using canine blood samples. R statistical language was employed for the statistical analyses. A major hemoglobin fraction (named HbA0) and a minor one (named HbA2) were identified in 100% and 47.4% of samples, respectively. The within-run and between-run CV was 0.1% for HbA0, and 9.1% and 11.2% for HbA2, respectively. The extremely narrow range of HbA0 and HbA2 values hampered a linearity study using canine blood samples. The RIs for HbA0 and HbA2 were 98.9–100% and 0–1.1%, respectively. HbA0 and HbA2 values were not significantly correlated with age (P = 0.866) or reticulocyte count (P = 0.731). No differences were observed in the median HbA0 and HbA2 between the two sexes (P = 0.887), and healthy and anemic dogs (P = 0.805). In conclusion, the capillary electrophoresis revealed a major hemoglobin fraction and an inconsistently present minor fraction. No effect of age, sex, anemia, or regenerative status of anemia was detected. The assay used was validated and RIs were generated, so as to be suitable for use in future investigations.

Partial Text

Hemoglobin is the oxygen-carrying moiety of erythrocytes. Structurally, it is a globular polypeptide tetramer, which consists of two pairs of unlike globin chains that form a shell around a central cavity. The latter contains four oxygen-binding heme groups, each of which is covalently linked to a globin chain.

The blood samples used in this study were aliquots of specimens collected (owners’ consent provided) for diagnostic purposes, routine health check, or pre-operatively from healthy dogs referred to the Companion Animal Clinic, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, Greece. The reference individuals were selected by a direct a priori method, based on the following inclusion criteria: age >6 months, up-to-date vaccination and deworming status, no history of illness or medication in the preceding month, and unremarkable physical examination and complete blood count. The distribution of canine breeds that were used in this study are presented in Table 1. Blood samplings were performed at admission by jugular venipuncture and the samples were collected into K3-ethylene diamine tetra-acetic acid (EDTA) coated 2mL tubes (Deltalab, Barcelona, Spain). Anemia was defined as red blood cell count <5.36 x 109/L, or hemoglobin concentration <122 g/L, or hematocrit <0.372 L/L [12]. The anemia was classified as regenerative when the absolute reticulocyte count was >60,000/μL [13]. The diagnoses for all anemic dogs included in the present study can be found in Table 2. Grossly hemolyzed (in vitro hemolysis) and lipemic samples were excluded from the study. A complete blood count was performed on the Advia 120 hematology analyzer (Siemens Healthcare Diagnostics, Deerfield, USA) within 2 h of sampling.

In total, 95 dogs were sampled. The reference population comprised 53 dogs (27 males and 26 females) with mean (±SD) age of 6.0±3.8 years, hemoglobin concentration of 155±16 g/L, and reticulocyte count of 31.1±15.5 x 106/L. The anemic population comprised 42 dogs (19 males and 23 females) with mean (±SD) age of 6.6±4.1 years, hemoglobin concentration of 75±27 g/L, and reticulocyte count of 179.5±158.4 x 106/L. The anemia was classified as non-regenerative in 16/42 (38.1%) dogs and regenerative in 26/42 (61.9%) dogs.

In this study, the electrophoretic pattern of canine hemoglobin was investigated using a new automated capillary electrophoresis assay, addressing a significant knowledge gap in hemoglobin research. This is the first study to date in which an automated assay has been validated for canine hemoglobin analysis and appropriate reference intervals have been calculated for adult dogs. The effect of age and sex on canine hemoglobin electrophoretic pattern was also evaluated. Finally, we investigated if anemias that were not attributed to a hemoglobin disorder, could affect the hemoglobin electrophoretic pattern.

The canine hemoglobin consists of a major fraction and a minor one, inconsistently present in very low proportions. A new automated capillary electrophoresis assay was validated for the separation of canine hemoglobin fractions and appropriate RIs were generated. Our study indicates no effect of age, sex or regenerative status of anemia on hemoglobin electrophoretic pattern among adult dogs, while no quantitative or qualitative hemoglobin abnormalities were detected in the anemic dogs without evidence for a hemoglobin disorder. The capillary electrophoresis assay used in this study is the only validated assay that can be used in future research studies on canine hemoglobin or in clinical cases suspected of having a hemoglobin disorder.