Journal of Avian Medicine and Surgery 22(2):108–113, 2008 ’ 2008 by the Association of Avian Veterinarians

Comparison of Hematologic Values in Blood Samples With or Dipotassium Ethylenediaminetetraacetic Acid in Hispaniolan Amazon Parrots (Amazona ventralis) David Sanchez-Migallon Guzman, Lcdo en Vet, Mark A. Mitchell, DVM, MS, PhD, Stephen D. Gaunt, DVM, PhD, Dipl ACVP, Hugues Beaufre`re, Dr Vet Med, and Thomas N. Tully, Jr, DVM, MS, Dipl ABVP (Avian) Dipl ECAMS

Abstract: Blood samples were collected from 20 Hispaniolan Amazon parrots (Amazona ventralis) and were divided into tubes that contained dipotassium ethylenediaminetetraacetic acid (K2EDTA) and lithium heparin. Complete blood cell counts were determined in each sample within 2 hours of collection. The level of agreement in results was moderate for plasma protein, packed cell volume (PCV), and leukocyte, monocyte, and lymphocyte counts between the anticoagulants. Plasma protein and PCV values were significantly lower in samples with lithium heparin than in those with K2EDTA, whereas lymphocyte numbers were significantly higher in lithium heparin samples than in K2EDTA samples. The level of agreement was good for the other cell types (heterophils, eosinophils, and ) when comparing the different anticoagulants. The poor level of agreement between anticoagulants with the increase in thrombocyte clumping in lithium heparin samples indicates that the use of lithium heparin as may affect thrombocyte count. No negative effects on morphology and staining of blood cells were apparent in smears from heparin samples compared with K2EDTA samples. Within the different values compared, the limits of agreement are small enough to be confident that lithium heparin can be used for routine CBC counts in a clinical setting. The use of the same anticoagulant should be recommended to follow trends within the same patient, especially when considering plasma protein concentration, PCV, and lymphocyte count. Key words: Anticoagulant, hematology, lithium heparin, dipotassium ethylenediaminetetraace- tic acid, avian, Hispaniolan Amazon parrots, Amazona ventralis

Introduction In domestic , K2EDTA is the recom- mended anticoagulant for storing blood samples Dipotassium ethylenediaminetetraacetic acid for hematologic testing. In many domestic species, (K EDTA) and lithium heparin are 2 of the most 2 heparin alters leukocyte morphology, provides commonly used anticoagulants in avian medicine. poorer staining quality to the cells, and does not ThemodeofactionofKEDTA is by binding 2 prevent aggregation.2,3 In birds, K EDTA calcium , which are essential in the 2 is also generally recommended by some investiga- cascade and for cell-to-cell interaction. Heparin tors as the anticoagulant of choice for hematologic binds to and accelerates the activity of antithrom- testing,4,5 although it was shown to cause progres- bin III, which inhibits the action of and sive hemolysis of the red blood cells (RBCs) in other proteases necessary for coagulation.1 ostrich (Struthio camelus), jackdaws (Corvus From the Department of Veterinary Clinical Sciences, monedula), ravens (Corvus corax), black curassows School of Veterinary Medicine, Louisiana State University, (Crax alector), black crowned cranes (Balearica Baton Rouge, LA 70803-8410, USA (Guzman, Gaunt, Tully); pavonina), gray crowned cranes (Balearica regu- the Veterinary Teaching Hospital, Ontario Veterinary College, lorum), hornbills (Tockus alboterminatus), and University of Guelph, Ontario N1G 2W1, Canada (Beaufre`re); brush turkeys (Alectura lathami).4 In those species and the Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois, 1008 W Hazelwood where this occurs, heparin is the preferred Dr, Urbana, IL 61802, USA (Mitchell). anticoagulant.4,5 Other investigators report a

108 GUZMAN ET AL—HEMATOLOGIC VALUES IN HEPARIN AND EDTA SAMPLES FROM PARROTS 109 preference for heparin for both complete blood Twenty Hispaniolan Amazon parrots that be- cell (CBC) counts and plasma biochemical assay6 longed to a resident colony at Louisiana State or observed that after 24 hours, blood samples University were used for this study. The birds were anticoagulated with EDTA degenerate more than fed a pelleted diet (Exact Breeding Formula, Kaytee other types of anticoagulated blood samples.7 Products Inc, Chilton, WI, USA) and had unlim- Only a few controlled studies compared the ited access to water. All birds were apparently hematologic values of avian blood samples healthy based on results of physical examination. collected in heparin and EDTA.1,8–10 In a study For , birds were manually re- that evaluated pigeon blood, the temporal effect strained, and a 1-ml blood sample was collected of different concentrations of heparin and dipo- from the right jugular vein of each bird with a 25- tassium salts on the was evaluated. gauge needle attached to a 3-ml plastic syringe. Results showed EDTA to be superior to heparin Blood samples were collected from 2 birds each in producing a constant hematocrit value as time day over a 10-day period, and the same person progressed and in the period of time before collected all blood samples (DSMG). This sched- hemolysis occurred in the blood.8 In a similar ule was followed to minimize the likelihood of the study, also in pigeons, the effects of different laboratory being rushed to process all of the anticoagulants in various concentrations on samples in a single day and to ensure that all hematocrit, osmolarity, and pH was studied, samples were processed within 2 hours of collec- and heparin was found to be the most suitable tion. Immediately after collection, each sample anticoagulant.9 Most recently, a study in macaws was separated into 2 tubes: 1 tube contained (Ara species) compared the temporal effects of 3 K2EDTA, and the second tube contained lithium commonly used anticoagulants. However, the use heparin (Microtainer, Becton Dickinson and of heparin-coated syringes in this study limited Company, Franklin Lakes, NJ, USA). The order the ability to fully ascertain the effect of of sample placement into the anticoagulant tubes individual coagulants.1 was alternated between birds. Blood samples were Comparable investigations were recently done stored at room temperature until processed within in reptiles. In studies that evaluated blood 2 hours of collection by the clinical pathology samples from green iguanas (Iguana iguana)11 laboratory at the Louisiana State University and yellow-blotched map turtles (Graptemys School of Veterinary Medicine. A single techni- flavimaculata),12 hematologic results from lithium cian who was blinded to animal number reviewed all samples. For each sample, packed cell volume heparin and K3-EDTA anticoagulated samples differed significantly, whereas, in another study, (PCV), plasma protein concentration, total leu- which evaluated Burmese pythons (Python mo- kocyte count, and differential leukocyte count lurus bivittatus), no significant differences were were determined. found between the sample results.1 In Hermann’s To determine PCV, 1 plain microhematocrit tube (Fisherbrand, Fisher Scientific, Pittsburgh, tortoises (Testudo hermanni), K3EDTA was shown to lower the PCV.13 Based on results of PA, USA) was filled from each anticoagulated these studies in reptiles, differences in hematologic sample and then centrifuged for 4 minutes at results between anticoagulants may be at the 1452g. The PCV to the nearest percentage was order or species level and not at the class level. read by using a microhematocrit chart. The tubes The purpose of this study was to measure the were then broken at the buffy coat, and the level of agreement and determine significant plasma protein concentration was determined by differences in the hematologic values of Hispan- refractometer (Leica Microsystems Inc, Buffalo, 14 iolan Amazon parrots (Amazona ventralis)when NY, USA). The total leukocyte concentration blood samples were placed in 2 different antico- was calculated by an indirect method, the agulants. We hypothesized that the level of eosinophil Unopette method (Unopette, Becton agreement would be good and that results of the Dickinson) and by hemacytometer (Improved CBC count would not differ significantly between Neubauer Hemacytometer, Bright-Line, Cam- bridge Instruments Inc, Buffalo, NY, USA). anticoagulants. The differential count was calculated from a Wright stained blood smear.15–17 The blood smear Materials and Methods was prepared by mixing 5 drops of the blood This study was performed according to the sample and 1 drop of 22% bovine albumin regulations established by the Louisiana State (Immucor Inc, Norcross, GA, USA). Total white University Animal Care and Use Committee. blood cell (WBC) counts were calculated by the 110 JOURNAL OF AVIAN MEDICINE AND SURGERY formula: (total WBC per 9 squares of both sides lithium heparin samples (Table 1). Heterophil of the hemacytometer) 3 1.1 3 16 3 100/ counts between the 2 anticoagulants were not (percentage of heterophils + eosinophils). Abso- significantly different (P 5 .08). Although the P lute leukocyte counts were then calculated by , .10, the observed power for the comparison multiplying this number with the percentages between the methods used for heterophil counts obtained from the modified Wright stained blood was .69. No significant differences were detected smears. The blood smears were evaluated for cell for the other hematologic parameters. The level of preservation by determining the presence of agreement between the anticoagulants for heter- smudged cells or free nuclei. The effect of the ophils (mean bias: 20.31; limits of agreement: anticoagulant on the cell-staining characteristics 21.64 to 1.01), eosinophils (mean bias: 0.03; was subjectively determined by evaluating the limits of agreement: 20.46 to 0.53), and basophils eosinophilic, basophilic, and metachromatic (mean bias: 20.02; limits of agreement: 20.60 to staining patterns of the leukocyte granules and 0.56) were good; whereas, the level of agreement the intensity of nuclear chromatin staining in the for PCV (mean bias: 23.5; limits of agreement: erythrocytes, leukocytes, or thrombocytes. The 27.06 to 0.07), WBC (mean bias: 0.98; limits of presence of thrombocyte clumping and cell agreement: 24.10 to 6.07), lymphocytes (mean morphology and staining characteristics were bias: 1.11; limits of agreement: 22.56 to 4.79), evaluated microscopically at 31000. monocytes (mean bias: 0.18; limits of agreement: The distribution of the hematologic data was 21.15 to 1.52), and plasma protein (mean bias: evaluated for normality by the Shapiro-Wilk test. 20.635; limits of agreement: 21.2 to 20.01) were The mean, SD, and minimum and maximum considered only moderate. values were reported for normally distributed There were small to large clusters of intact data, whereas the median, deciles D1 and D9, and thrombocytes in the monolayer area of the blood minimum and maximum values were reported for smears in 65% (13/20) of the K2EDTA samples and non-normally distributed data. Nonparametric 95% (19/20) of the lithium heparin samples; data were log transformed for parametric analysis however, this difference was not significant (P 5 (eosinophil and counts). A paired .35). Five percent (1/20) of the birds had no sample t test was used to determine if differences thrombocyte clumping in either EDTA or heparin occurred between the 2 anticoagulants for con- samples. Sixty percent (12/20) of the birds had tinuous variables, whereas the Fisher exact test thrombocyte clumps in both EDTA and heparin was used to evaluate the same comparison samples. The k statistic for the comparison of between categorical data (thrombocyte clump- thrombocyte clumping between the 2 anticoagu- ing). Statistical significance was set at P # .05. A lants was 0.18. Subjectively, there were no apparent post hoc power was calculated if P , .10. Data differences in cell preservation and staining char- were analyzed by using SPSS 11.0 (SPSS Inc, acteristics between the anticoagulants. Chicago, IL, USA). The level of agreement between samples was Discussion determined by the Bland-Altman method. For In this study, the hematologic values in blood this analysis, bias was defined as the mean samples from Hispaniolan Amazon parrots col- difference between results of the 2 methods, and lected in lithium heparin and in K EDTA were the limits of agreement were calculated as mean 2 compared. The level of agreement between the bias 6 1.96 3 SD. MedCalc (MedCalc Software, samples was lowest for the plasma protein Mariakerke, Belgium) was used to analyze the concentration, PCV, and lymphocyte count, and data. For categorical data, the Cohen k test was were highest for the remaining blood cell types. used to measure agreement. The limits of agreement in the results of the different values compared are subjectively small Results enough for us to be confident that lithium heparin Results showed significant differences between can be used for routine complete blood counts in the 2 anticoagulant samples in PCV measure- a clinical setting. The same anticoagulant should ments (P , .001), plasma protein concentration be used to follow trends within the same patient, (P , .001), and lymphocyte counts (P , .05). especially when considering plasma protein, PCV, Plasma protein and PCV values were significantly and lymphocyte count. lower in lithium heparin samples, whereas lym- Regression or correlation coefficients have been phocyte counts were significantly higher in used historically to measure the level of agreement GUZMAN ET AL—HEMATOLOGIC VALUES IN HEPARIN AND EDTA SAMPLES FROM PARROTS 111

for a continuous biologic parameter; however, ium 1.0

a 18–21

l) neither of these methods is optimal. The 2 m / 1.1) 3 technique considered most appropriate for mea- 0.0-0.5 0.0 2 10 suring the level of agreement is the Bland-Altman 6 6 3 Basophils ( (0.0–1.0) (0.0 method and was the technique used in this 0.1 0.2 study.19,21 The level of agreement between the 2 methods is limited by the precision (ie, repeatabil- 0.6 0.7 a ity of the results) of the methods compared. If 1 l) 2 2 m 1.0) 1.0) / method has poor precision, the agreement be- 3 0.0 0.0 2 2 19

10 tween the 2 methods will likely be poor. The 6 6 3 ( (0.0 (0.0

Eosinophils Unopette system is a standardized method, with a

0.1 0.1 known coefficient of variation (6.8%), for deter- mining the leukocyte concentration.22 There are

l) no other reports regarding precision for other 0.5 0.4 m / 2.3) 1.8)

3 hematologic values (eg, PCV, plasma protein) 6 2 6 2 10 calculated in this study. 3 ( (0.3 (0.3 1.1 1.0 Monocytes The values for PCV were significantly lower when using lithium heparin than with K2EDTA. These differences could not be attributed to l) 2.0 1.6 m hemolysis, because hemolysis was not appreciated / 8.1) 6.8) 3 2 2 6 6 in any of the anticoagulated blood samples of our 10 b b 3

( study. In studies of blood samples, (1.4 (1.4 3.6 4.5 Lymphocytes K2EDTA outside of the optimum concentration (1–2 mg/ml blood) causes a progressive decrease

eosinophils, basophils]) for hematologic values by anticoagulant, lith in the estimate of PCV values.23,24 The effect of L)

1.8 1.6 K EDTA is attributed to the reduction of RBC

m 2- 7.4) 6.8) / 3

6 2 6 2 volume caused by the anticoagulant cation in the 10 23

3 plasmatic osmotic pressure. Heparin alone, ( (2.3 (1.4 5.2 5.0 Heterophils however, causes little alteration in corpuscular size and is considered a more suitable anticoag-

20). ulant, because varying the concentration has little l) 5 23,24 3.1 2.9

m effect on the PCV values. In our study, the 15.9) 14.4) / 3 2 2 6 6 tubes were filled to an appropriate volume 10 3 (5.8 (5.1 ( according to manufacturer instructions; however, cell count 10.4 10.9 White blood the use of lithium heparin, instead of heparin, EDTA) (N

2 could account for the difference in results. Plasma protein values were significantly lower ) % 3.8 3.5 in samples anticoagulated with lithium heparin 60.0) 57.0) 2 2 6 6 than in those with K2EDTA. The use of EDTA b b can affect the refractometric reading of pro- Packed cell volume ( (47.0 (46.0 25,26 53.0 50.3 teins, whereas similar effects have not been reported with heparin. In this study, total protein values were estimated by refractometry, approx- imating the total solids measured. The accuracy 0.5 0.3 6.5) 6.0)

6 2 6 2 of the refractometric method to determine total (g/dl) b b Plasma protein protein concentration in avian blood is considered (4.9 (4.0 poor.27 Because of the higher glucose and lower total protein concentrations in birds, correlation of results from the refractometer and the biuret methods may not be possible in some species.28 Refractometry should be considered a rapid

Descriptive measures (mean, SD, and range; or median and deciles D1 and D9 [ method for determining an estimate of the fluid

.05. protein. Ideally, total protein concentrations , -EDTA 5.7 should be determined by plasma protein electro- Anticoagulant 2 P Median and deciles D1 and D9. Lithium heparin 5.2 Table 1. heparin, or dipotassium ethylenediaminetetraacetic (K K a b phoresis. 112 JOURNAL OF AVIAN MEDICINE AND SURGERY

In this study, no significant differences were Although K3EDTA was used in a study 1 observed in the total leukocyte count between conducted in macaws, K2EDTA was used in anticoagulants. However, the use of heparinized our study. The differences in these 2 formulation blood samples has been considered to result in were studied in human medicine, with the erroneous counts because of leukocyte and conclusion that the differences observed by using thrombocyte clumping and improper staining of K3EDTA glass tubes versus K2EDTA plastic cells.29 In 1 study in macaws (Ara species), no tubes were unlikely to be of any clinical signifi- significant differences were found in results from cance.32 lithium heparin samples at 3 and 24 hours after Subjectively, no negative effects on morpholo- sampling, whereas, at 12 hours, WBC and RBC gy and staining of blood cells were apparent in lysis were markedly increased in K3EDTA- smears from heparin samples compared with 1 anticoagulated samples. Because heparin-coated K2EDTA samples. The poor level of agreement syringes were used in that study, however, the between anticoagulants with the increase in effect of the individual coagulants could not be thrombocyte clumping in lithium heparin samples fully ascertained. In several reptile species, indicates that the use of lithium heparin as an significantly higher values for total WBC counts anticoagulant may affect thrombocyte count. The and most differential leukocyte counts were found limits of agreement within the remaining values in EDTA samples,11,12 whereas, in another study, that were compared are small enough to be there were no differences in results with different confident that lithium heparin can be used for anticoagulants.1 routine CBCs in Hispaniolan Amazon parrots The leukocyte counts were determined by an when the samples are processed within 2 hours. indirect method with phloxine B solution. Phlox- To follow trends within the same patient, the ine B stains only the acidophilic same anticoagulant should be used for serial (heterophils and eosinophils). Basophils can also comparisons, especially when considering plasma be identified by using increased contrast when protein concentration, PCV, and lymphocyte counting the leukocytes. The count was corrected count. Further studies are needed to evaluate for mononuclear cells once the differential was the temporal effect of anticoagulants on the made. The slight decrease in the hematologic values in avian species. numbers in lithium heparin samples, which would result in an overestimate of the mononuclear cells, References could explain the higher lymphocyte count in those samples. Otherwise, no significant differ- 1. Harr KE, Raskin RE, Heard DJ. Temporal effects ences were found in heterophil, monocyte, or of 3 commonly used anticoagulants on hematolog- basophil numbers in the samples stored in ic and biochemical variables in blood samples from macaws and Burmese pythons. Vet Clin Path. K EDTA or heparin. 2 2005;34:383–388. Heparin is reported to alter the nuclear staining 2. Knoll JS. Clinical automated hematology sys- of blood cells and to cause changes in avian blood tems. In: Feldman BF, Zinkl JG, Jain NC, eds. 30 cell morphology. This widely quoted study Schalm’s Veterinary Hematology, 5th ed. Philadel- stated that heparin has a negative effect on phia, PA: Lippincott, Williams, & Wilkins; morphology and staining was based on blood 2000:3–11. samples from domestic chickens that were citrat- 3. Jain NC. Hematologic techniques. In: Jain NC, ed. ed, washed, and subsequently exposed to high Veterinary Hematology. Philadelphia, PA: Lea and concentrations of heparin and that were read Febiger; 1986:23–86. within 8 hours.31 In our study, these changes were 4. Samour J. Diagnostic value of hematology. In: not evident on blood-smear examinations from Harrison GJ, Lightfoot TL, eds. Clinical Avian heparin samples when compared with those from Medicine. Palm Beach, FL: Spix Publishing; EDTA samples. Also, samples in our study were 2006:587–610. 5. Campbell TW. Avian Hematology and Cytology. processed within 2 hours. Further studies are 2nd ed. Ames, Iowa: Iowa State University Press; needed to characterize the temporal effects of 1995. individual anticoagulants. Thrombocyte clumping 6. Lumeij J. Avian clinical pathology. Vet Q. was increased in the lithium heparin samples, and 1987;9:249–254. the agreement was poor with K2EDTA samples, 7. Fudge AM. Avian . In: indicating that the thrombocyte count may be Fudge AM, ed. Laboratory Medicine, Avian and affected by the choice of anticoagulant. However, Exotic Pets. Philadelphia, PA: WB Saunders; a thrombocyte count was not done in this study. 2000:9–18. GUZMAN ET AL—HEMATOLOGIC VALUES IN HEPARIN AND EDTA SAMPLES FROM PARROTS 113

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