Relationship of hemoglobin concentration to packed cell volume in avian blood samples.
Abstract: To determine whether a proportional relationship exists between hemoglobin (Hgb) concentration and pack cell volume (PCV) in avian blood samples, Hgb concentration measured by a point-of-care portable hemoglobinometer and corresponding PCV were determined in blood samples collected from 128 birds comprising 13 avian orders. For all birds evaluated and pooled across orders, a significant and consistent relationship was found between Hgb concentration and PCV, expressed as Hgb = 0.304 x PCV + 0.461. To determine whether the relationship of PCV and Hgb differs, 9 avian orders with n [greater than or equal to] 8 samples per order were analyzed by linear regression. Individual slopes for the 9 orders did not differ significantly (P = .44), indicating that a single slope can be used to model the avian Hgb to PCV relationship for these taxonomic orders. A single intercept can also be used, with the exception of order Phoenicopteriformes, which was the only intercept that was significantly different from 0 (P = .01). These results indicate that a simplified relationship of Hgb (g/dL) = 0.30 x PCV provides a reasonable estimate of Hgb concentration from the PCV of birds from the orders Anseriformes, Columbiformes, Falconiformes, Galliformes, Passeriformes, Psittaciformes, Sphenisciformes, and Strigiformes, but a separate relationship of Hgb = 0.217 x PCV + 6.69 might be warranted for the order Phoenicopteriformes.
Key words: blood, packed cell volume, hemoglobin, hemoglobinometer, Anseriformes, Columbiformes, Falconiformes, Galliformes, Passeriformes, Psittaciformes, Sphenisciformes, Strigiformes, Phoenicopteriformes, avian
Evaluation of the erythron is a commonly used diagnostic technique to assess anemia in mammalian veterinary patients. Ideally, complete assessment of erythrocytes includes evaluation of the packed cell volume (PCV), total red blood cell (RBC) count, hemoglobin (Hgb) concentration, calculated RBC indices, reticulocyte count, and RBC morphology. (1-3) Accurate measurement of the Hgb concentration in avian blood by the traditional cyanomethemoglobin technique requires lysis of erythrocytes followed by centrifugation to remove free nuclei. (1,3,4) Many commercial clinical pathology laboratories do not measure Hgb concentrations and do not provide RBC indices for avian blood samples on a complete blood cell (CBC) count? Therefore, avian anemias are commonly characterized on the basis of PCV and RBC morphology alone. (5,6)
Small, portable, point-of-care hemoglobinometers are commercially available that use a spectrophotometric double-wavelength azide-methemoglobin method to correct for sample turbidity resulting from lipid particles, cell stromata, and large proteins that tend to interfere with the cyanomethemoglobin method. (7) These portable hemoglobinometers are frequently used to calculate the Hgb concentration in outpatient settings in human medicine and have been demonstrated to provide reliable results in veterinary patients, including reptilian and avian species. (7-11) This technology provides an opportunity to evaluate a second quantitative parameter rapidly, in addition to the PCV for avian blood samples.
In mammals, the normal Hgb concentration is generally considered to be approximately one-third of the PCV. (3,12,13) Alteration of this proportion can help to characterize anemia and is reflected in changes in the RBC indices. A similar relationship between PCV and Hgb concentration in avian blood has been assumed, but no studies have confirmed this assumption. This study was undertaken to determine whether a consistent relationship exists between the PCV and Hgb concentration in avian blood samples and, if so, whether the relationship is similar among the avian taxonomic orders included in the study.
Materials and Methods
A wide range of captive and free-ranging birds are regularly presented to the Zoological, Exotic, and Wildlife Medicine Service at Oklahoma State University and to the Animal Health Department at the Tulsa Zoo (Oklahoma). Data from birds identified with chronic or longstanding illnesses were excluded from this study. Blood samples (0.25-0.5 mL, all volumes within recommended limits for collection tube size) were opportunistically obtained from 128 birds by jugular or basilic venipuncture and placed in lithium heparin vials (BD Microtainer tubes, BD Vacutainer Systems, Franklin Lakes, NJ, USA). A PCV was obtained for each sample after centrifugation (5 minutes at 10 400 rpm) of a glass microhematocrit tube (75 mm x 1.1-1.2 mm diameter). The remaining blood samples were frozen (-20[degrees]C [-4[degrees]F], frost free) in the lithium heparin tubes for up to 4 weeks before analysis. Preceding evaluation, the thawed samples were gently agitated by hand or by test tube mixer (Fisher Healthcare, Houston, TX, USA), for up to 5 minutes. Samples that did not return to a liquid suspension were not included in the study.
The Hgb concentration of batched samples was analyzed with the use of a portable hemoglobinometer (HemoCue 201+ analyzer, HemoCue Inc, Lake Forest, CA, USA). The blood sample was drawn into a cuvette by capillary action, and erythrocyte membranes were lysed by sodium deoxycholate. The iron in the freed Hgb was oxidized from the ferrous to the ferric state and combined with azide to form azide-methemoglobin within the cuvette. The hemoglobinometer measured absorbance at both 565 nm for methemoglobin and 880 nm to determine the degree of sample turbidity. Duplicate determinations of Hgb concentration were made on each sample, and average values were reported.
To determine variation in measurements and the effects on the calculated Hgb concentration of freezing, storage for up to 4 weeks, and thawing, blood samples were obtained by jugular venipuncture from 3 birds (a golden eagle [Aquila chrysaetos], a red-tailed hawk [Buret jamaicensis], and a Canada goose [Branta canadensis]) on 2 separate occasions and from 2 birds (a pigeon [Columba livia] and an eclectus parrot [Eclectus roratus]) on 1 occasion, then placed in lithium heparin tubes. A total of 15 blood samples resulted from 7 blood samples being split into equal volumes, each >0.25 mL; an eighth blood sample was insufficient in volume to be split. Each of the 15 fresh heparinized samples was placed in a microhematocrit tube and centrifuged as previously described. The PCV of each sample was measured, and the respective Hgb concentration was determined by hemoglobinometer. The 15 heparinized blood samples were then frozen (-20[degrees]C [-4[degrees]F]) and stored for 3-4 weeks. Samples were thawed, and the respective postfreezing Hgb concentrations were reanalyzed as a batch by the same investigator.
Paired t tests were used to compare the Hgb concentrations in the split samples of the fresh heparinized blood; in the split samples of blood that had been frozen, stored, and thawed; and for all fresh samples with those that had been frozen, stored, and thawed. Linear regression analysis was used to determine the relationship of PCV to Hgb concentration of the pre- and postfreezing heparinized samples, as well as to compare the relationship of PCV to Hgb concentration in blood samples from all birds. Separate linear regression analyses were used to compare the relationship of Hgb concentration to PCV among the taxonomic orders of birds with n [greater than or equal to] 8 samples in the study. The slopes and intercepts of these 2 regressions were compared with the use of an indicator variable model. All statistical analyses were conducted by PC SAS Version 8.2 software (SAS Institute, Cary, NC, USA) and included calculation of the mean and standard deviation (SD) for data with [greater than or equal to]5 samples from the same species. The minimum alpha level for statistical significance was P [less than or equal to] .05.
A total of 128 blood samples from 13 avian orders comprised of 33 species were used in the study (Table 1). Linear regression analysis of the combined data for all birds in all avian orders revealed a significant relationship (P < .001) between the PCV and Hgb concentration described as Hgb (g/dL) = 0.304 X PCV + 0.461, with a correlation coefficient of [r.sup.2] = .827 (Fig 1).
In an effort to ascertain whether this model was appropriate for all avian orders, separate linear regression analyses were performed for the 9 avian orders with n [greater than or equal to] 8 samples and did not reveal significant differences (P = .44) in the 9 slopes of the Hgb to PCV relationship (Fig 2). Furthermore, the intercepts associated with the regressions for 8 of the 9 avian orders tested were not significantly different (P > .05) from zero, but the intercept associated with the regression for Phoenicopteriformes was significantly different (P < .01) from zero at +6.69. The individual Hgb to PCV regression equations for the 9 avian orders are presented in Table 2. The separate relationship for Phoenicopteriformes was described as Hgb = 0.217 x PCV + 6.692 with [r.sup.2] = .793 (P < .003). The use of respective intercepts for each of the 9 avian orders with a common slope of 0.297 provided a simplified relationship of Hgb = 0.297 x PCV, with P = .001 and [r.sup.2] = .918 for all 122 birds in the 9 orders analyzed.
Within the smaller study, to determine whether freezing and storage affected Hgb values, no significant differences were found in the Hgb concentrations in the 7 split samples of fresh heparinized blood (P = .76); the means [+ or -] SD were 14.5 [+ or -] 2.1 g/dL and 14.5 [+ or -] 2.2 g/dL, respectively. No significant difference in Hgb concentrations from these 7 split samples was found after freezing, storage, and thawing (P = .79); the means [+ or -] SD were 14.4 [+ or -] 2.1 g/dL and 14.4 [+ or -] 2.3 g/dL, respectively. The Hgb concentrations obtained on all 15 samples of fresh heparinized blood and in these samples after freezing, storage, and thawing were combined, respectively, and no significant difference (P = .49) was found in Hgb concentrations before and after freezing; means [+ or -] SD were 14.7 [+ or -] 2.2 g/dL and 14.7 [+ or -] 2.3 g/dL, respectively. The linear relationship for PCV to Hgb concentration in fresh heparinized blood samples was described as PCV = 2.99 x Hgb concentration + 2.4 ([r.sup.2] = .98), and after freezing, storage, and thawing as PCV = 2.91 x Hgb concentration + 3.7 ([r.sup.2] = .97), respectively. The slopes and intercepts of these 2 linear relationships were not significantly different (P = .68 and P = .73, respectively).
[FIGURE 1 OMITTED]
[FIGURE 2 OMITTED]
Hemoglobin in birds (as in mammals) functions as a tetramer, which, in most avian species, is composed of combinations of 2 types of [alpha]-heme subunits with consistent [beta] subunits. (14) Alterations in the normal relationships among PCV, Hgb, and RBC size are routinely used as calculated RBC indices to better characterize hematopathology in mammalian patients. (3,15) Anemia with altered iron and Hgb metabolism has been associated with heavy metal toxicoses in birds. (16) The concentration of Hgb in both mammalian and avian blood samples has been traditionally measured by the cyanomethemoglobin method. (1,3,4,17) Commercial laboratory analytical protocols often do not provide measurement of Hgb levels in species with nucleated erythrocytes or provide for calculation of the corresponding RBC indices. (5) With the availability of point-of-care hemoglobinometers that are able to compensate for nucleated erythrocytes by double wavelength spectrophotometry, obtaining Hgb levels from small volumes of avian blood is possible in less time than is required to centrifuge a PCV, therefore obtaining useful information for characterizing anemias. (8,11)
The limited volumes of blood that practically can be collected from small and easily stressed birds can complicate clinical evaluation of avian patients. (1,6) Small sample volumes can result in a limited number of diagnostic tests performed on a single sample. The HemoCue analyzer cuvettes require 20 [micro]L of blood, which is approximately half the volume required to fill a short (4.3-cm) microcapillary tube. (7) The small blood volume that can be accommodated by the HemoCue makes evaluation of Hgb a reasonable supplement to the PCV for routine avian hemograms. The consistent relationship between PCV and Hgb in birds with no evidence of chronic or metabolic disease, as established in this study, would also permit the use of Hgb level in place of a PCV in cases of extremely limited blood volumes.
The nearly identical Hgb concentrations among split samples of fresh heparinized blood and among those split samples after freezing, storage, and thawing, suggests that Hgb concentrations obtained by the HemoCue on either fresh or frozen blood are repeatable. In addition, the Hgb concentrations obtained on freshly drawn heparinized blood samples were also nearly identical to those obtained on these samples after being frozen, stored, and thawed, indicating no significant alteration in Hgb concentration in the event that it is necessary to freeze and store samples before analysis. The linear relationship of the PCV to Hgb concentration of fresh heparinized blood samples and that of the same samples after being frozen, stored, and thawed were also very similar, with no significant difference in either the slopes or intercepts, which supports the use of frozen samples in this study.
Because birds sampled in this study were either clinically normal or had evidence of acute illness
or trauma only, most PCV values obtained fell roughly within reference ranges, with relatively few samples representing either severe anemia or polycythemia. Therefore, the relationship between Hgb concentration and PCV might have differed had more values outside of reference ranges been included. However, the 3 avian orders represented by the widest PCV ranges in this study (Falconiformes, Passeriformes, and Sphenisciformes) had calculated regression equations similar to those with more narrow PCV ranges.
A strong linear relationship of Hgb to PCV was found for birds in general, with good agreement of this relationship among 9 avian orders. Because the individual slopes for the 9 orders compared were not significantly different, a single slope can be used to model the relationship of Hgb to PCV, regardless of taxonomic order. With the possible exception of Phoenicopteriformes, a single intercept can also be used for all avian orders. Using the respective intercepts for each of the 9 orders with [greater than or equal to]8 samples in this study and the combined common slope of 0.297, we provide a model for Hgb concentration estimates from the PCV of blood samples with a significance of P < .001 and [r.sup.2] = .918.
On the basis of the Findings of this study, Phoenicopteriformes might have a different relationship between Hgb and PCV, with a lower linear regression slope (0.217) and larger intercept (+6.692) compared with those determined for the other 8 orders evaluated. The reason for this remains unknown but could reflect the small sample size from this order, and from American flamingoes (Phoenicopterus ruber) specifically, and the absence of samples from individuals with anemia. Further investigation might be warranted to confirm whether the Hgb to PCV relationship of Phoenicopteriformes is different from that of the other avian orders examined in our study and, if so, why.
The findings of this study demonstrate that reasonable estimates of Hgb concentrations in avian blood samples can be made on the basis of PCV determinations and vice versa. We propose that the simplified relationship of Hgb = 0.30 x PCV, as extrapolated from both the equations determined for all birds sampled (slope = 0.304) and for the combined equations from the separate avian orders (slope = 0.297), provides a reasonable estimate of Hgb concentration from the respective PCV for blood samples from the orders Anseriformes, Columbiformes, Falconiformes, Galliformes, Passeriformes, Psittaciformes, Sphe nisciformes, and Strigiformes, in which the intercepts were not significantly different from zero. A separate relationship of Hgb = 0.217 x PCV + 6.69 might be warranted for the order Phoenicopteriformes.
Acknowledgments: Funding for this project was provided by the College of Veterinary Medicine at Oklahoma State University. We thank Dr Tim Georoff and Dr Kay Backues for their assistance in obtaining and contributing blood samples for this project.
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Karen E. Velguth, DVM, Mark E. Payton, PhD, and John P. Hoover, MS, DVM, Dipl ABVP, Dipl ACVIM Abstract: To determine whether a proportional relationship exists between hemoglobin (Hgb)
From the Department of Veterinary Pathobiology, College of Veterinary Medicine, Center for Veterinary Health Sciences (Velguth, Hoover) and the Department of Statistics, College of Arts and Sciences (Payton), Oklahoma State University, Stillwater, OK 74078, USA. Present address (Velguth): IDEXX Laboratories, 6100 East Shelby Drive, Memphis, TN 38141, USA.
Table 1. Species represented in the study evaluating the relationship of hemoglobin to packed cell volume in birds. Order Common name Scientific name Anseriformes Pekin duck Anas platyrhynchos domestica Canada goose Brama canadensis Mallard Anas platyrhynchos Ciconiiformes Great blue heron Ardea herodias Columbiformes Rock pigeon Columba livia Cuculiformes Greater roadrunner Geococcyx californianus Falconiformes Bald eagle Haliaeetus leucocephalus Golden eagle Aquila chrysaetos Broad-winged hawk Buteo platypterus Cooper's hawk Accipiter cooperii Red-shouldered hawk Buteo lineatus Red-tailed hawk Buteo jamaicensis American kestrel Falco sparverius Galliformes Domestic chicken Gallus gallus Ring-necked pheasant Phasianus colchicus Domestic turkey Meleagris gallopavo Gruiformes Common gallinule Gallinula chloropus Passeriformes Red-winged blackbird Agelaius phoeniceus Blue jay Cyanocitta cristata Plush-crested jay Cyanocorax chrysops American robin Turdus migratorius American crow Corvus brachyrhynchos European starling Sturnus vulgaris Pelicaniformes White pelican Pelecanus erythrorhynchos Phoenicopteriformes American flamingo Phoenicopterus ruber Psittaciformes Sun conure Aratinga solstitialis Green-winged macaw Ara chloroptera Hyacinth macaw Anodorhynchus hyacinthinus African grey parrot Psittacus erithacus Thick-billed parrot Rhynchopsitta pachyrhyncha Sphenisciformes Black-footed penguin Spheniscus demersus Strigiformes Barred owl Strix varia Great horned owl Bubo virginianus Number of Order Common name samples (n) Anseriformes Pekin duck 13 Canada goose 1 Mallard 1 Ciconiiformes Great blue heron 1 Columbiformes Rock pigeon 13 Cuculiformes Greater roadrunner 2 Falconiformes Bald eagle 1 Golden eagle 2 Broad-winged hawk 1 Cooper's hawk 3 Red-shouldered hawk 5 Red-tailed hawk 10 American kestrel 5 Galliformes Domestic chicken 5 Ring-necked pheasant 1 Domestic turkey 2 Gruiformes Common gallinule 1 Passeriformes Red-winged blackbird 1 Blue jay 2 Plush-crested jay 1 American robin 2 American crow 3 European starling 1 Pelicaniformes White pelican 2 Phoenicopteriformes American flamingo 8 Psittaciformes Sun conure 2 Green-winged macaw 2 Hyacinth macaw 4 African grey parrot 1 Thick-billed parrot 2 Sphenisciformes Black-footed penguin 15 Strigiformes Barred owl 11 Great horned owl 4 Table 2. Individual regression equations demonstrating the relationship of hemoglobin (Hgb) concentration to packed cell volume (PCV) for 9 avian orders and for all birds. Regression equation: Hgb (g/dL) = slope (a) x PCV (%) [+ or -] n intercept (b) P Avian orders Anseriformes 15 Hgb = 0.31 x PCV - 0.12 <.001 Phoenicopteriformes 8 Hgb = 0.22 x PCV + 6.69 (b) <.003 Columbiformes 13 Hgb = 0.28 x PCV + 1.94 <.001 Falconiformes 27 Hgb = 0.28 x PCV + 0.49 <.001 Galliformes 8 Hgb = 0.27 x PCV + 1.27 <.001 Passeriformes 10 Hgb = 0.33 x PCV + 0.11 <.001 Psittaciformes 11 Hgb = 0.34 x PCV - 0.10 <.001 Sphenisciformes 15 Hgb = 0.28 x PCV + 0.04 <.001 Strigiformes 15 Hgb = 0.37 x PCV - 1.47 <.001 All birds 122 Hgb = 0.30 x PCV + 0.46 <.001 [r.sup.2] Avian orders Anseriformes 0.88 Phoenicopteriformes 0.79 Columbiformes 0.83 Falconiformes 0.87 Galliformes 0.97 Passeriformes 0.94 Psittaciformes 0.81 Sphenisciformes 0.93 Strigiformes 0.88 All birds 0.83 (a) No significant differences (P > .05) were found in slopes among the 9 orders. (b) Although intercepts differed significantly (P > .05) among the 9 orders, only Phoenicopteriformes was significantly different from zero at P < .01.
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|Title Annotation:||Original Studies|
|Publication:||Journal of Avian Medicine and Surgery|
|Date:||Jun 1, 2010|
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