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Relationship of hemoglobin concentration to packed cell volume in avian blood samples.

Karen E. Velguth, Mark E. Payton, John P. Hoover,

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

Introduction

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.

Results

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]

Discussion

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.

References

(1.) Campbell TW, Ellis CK. Hematology of birds. In" Campbell TW, Ellis CK, eds. Avian and Exotic Animal Hematology and Cytology. 3rd ed. Ames, IA: Blackwell; 2007:3-50.

(2.) Fudge AM. Avian complete blood count. In: Fudge AM, ed. Laboratory Medicine." Avian and Exotic Pets. Philadelphia, PA: WB Saunders; 2000: 9-18.

(3.) Stockham SL, Scott MA. Erythrocytes. In: Stockham SL, Scott MA, eds. Fundamentals of Veterinary Clinical Pathology. 2nd ed. Ames, IA: Blackwell; 2008:107-221.

(4.) Pierson FW. Laboratory techniques for avian hematology. In" Feldman BF, Zinkl JG, Jain NC, et al, eds. Schalm's Veterinary Hematology. 5th ed. Baltimore, MD: Lippincott Williams & Wilkins; 2000:1145-1146.

(5.) Morrisey JK. Avian hematology. Proc Annu West Vet Conf [CD-ROM]. 2004.

(6.) Jones MP. Avian clinical pathology. Vet Clin North Am Exotic Anim Pract. 1999;2:663-687.

(7.) von Schenck H, Falkensson M, Lundberg B. Evaluation of "HemoCue," a new device for determining hemoglobin. Clin Chem. 1986;32: 526-529.

(8.) Chevalier H, Posner LP, Ludders JW, et al. Accuracy and precision of a point-of-care hemoglobinometer for measuring hemoglobin concentration and estimating packed cell volume in horses. J Am Vet Med Assoc. 2003;223:78-83.

(9.) Callan MB, Giger U, Oakley DA, et al. Evaluation of an automated system for hemoglobin measurement in animals. Am J Vet Res. 1992;53: 1760-1764.

(10.) Magona JW, Walubengo J, Anderson I, et al. Portable haemoglobinometers and their potential for penside detection of anaemia in bovine disease diagnosis: a comparative evaluation. Vet J. 2004; 168:343-348.

(11.) Gargan C, Arnold J. Comparison of two methods to determine hemoglobin in mammalian, reptilian, and avian blood (Poster C-l). Proc Annu ConfAm Soc Vet Clin Pathol. 2004:105.

(12.) Andrews GA, Smith JE. Iron metabolism. In: Feldman BF, Zinkl JG, Jain NC, et al, eds. Schalm's Veterinary Hematology. 5th ed. Balti more, MD: Lippincott Williams & Wilkins; 2000: 129-134.

(13.) Lassen ED, Weiser G. Laboratory technology for veterinary medicine. In: Thrall MA, Baker DC, Campbell TW, et al, eds. Veterinary Hematology and Clinical Chemistry. Philadelphia, PA: Lippincott Williams & Wilkins; 2004:3-37.

(14.) Knapp JE, Oliveira MA, Xie Q, et al. The structural and functional analysis of the hemoglobin D component from chicken. J Biol Chem. 1999; 274:6411-6420.

(15.) Thrall MA. Classification of and diagnostic approach to anemia. In: Thrall MA, Baker DC, Campbell TW, et al, eds. Veterinary Hematology and Clinical Chemistry. Philadelphia, PA: Lippincott Williams & Wilkins; 2004:83-88.

(16.) Christopher MM, Shooshtari MP, Levengood JM. Assessment of erythrocyte morphologic abnormalities in mallards with experimentally induced zinc toxicosis. Am J Vet Res. 2004;65:440446.

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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
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Geographic Code:1USA
Date:Jun 1, 2010
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