Hematologic values in healthy gyr x peregrine falcons (Falco rusticolus x Falco peregrinus).
Peregrine falcon (Health aspects)
Peregrine falcon (Research)
Lloyd, Christopher G.
|Publication:||Name: Journal of Avian Medicine and Surgery Publisher: Association of Avian Veterinarians Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2009 Association of Avian Veterinarians ISSN: 1082-6742|
|Issue:||Date: June, 2009 Source Volume: 23 Source Issue: 2|
|Topic:||Event Code: 310 Science & research|
|Geographic:||Geographic Scope: United Arab Emirates Geographic Code: 7UNIT United Arab Emirates|
Abstract: The gyr x peregrine (Falco rusticolus x Falco peregrinus)
hybrid is the most common falcon treated at Nad Al Shiba Veterinary
Hospital in Dubai. To improve the health assessment of individual birds
in the population treated at this hospital, we established reference
ranges of hematologic values for this hybrid falcon. The hematologic
parameters of 96 gyr x peregrine falcons were assessed and compared with
other published values for the same hybrid falcon.
Key words: hematology, reference values, avian, falcon, gyr, peregrine, Falco rusticolus, Falco peregrinus
The gyr x peregrine (Falco rusticolus x Falco peregrinus) hybrid currently is one of the most common falcons used by falconers in the United Arab Emirates and was the most frequently presented falcon to the Nad Al Shiba Veterinary Hospital in Dubai during August 2005 to January 2008. To more accurately assess the health status of these birds, the decision was made to establish in-house hematologic reference ranges for this hybrid falcon.
The importance of each laboratory establishing its own reference ranges by sampling a healthy population of the animals it serves has been established. (1,2) Hematologic parameters can be affected by a range of factors, (3) such as method of storage, (4-6) analysis, (7-10) stress on the animal, 11,12 and the age and sex of patients. (13-16) Published hematologic values exist for several falcon species, including saker (Falco cherrug), (1,17,18) gyr, (18,19) and peregrine falcons. (1,15,18) Two reports document the hematologic reference values of gyr x peregrine hybrids, (18,20) however, the methodology and results of these 2 accounts vary.
The purpose of this study was to establish hematologic reference ranges for healthy gyr x peregrine hybrids presented to Nad Al Shiba Veterinary Hospital for routine health screening. In-house laboratory hematologic methods were used for this purpose.
Materials and Methods
Blood samples were collected from gyr x peregrine falcons presented for routine health checks at the Nad Al Shiba Veterinary Hospital. Only healthy birds were included in the study. A bird was considered healthy based on these criteria: no abnormalities were detected on physical examination; results of bilateral endoscopic examination of the cranial, caudal, thoracic, and abdominal air sacs, and endoscopic examination of the trachea and crop were unremarkable; results of parasitologic examination of the crop and feces were normal. Of a total population of 96 gyr x peregrine falcons, 56 were female, 20 were male, and 20 falcons could not be reliably sexed by visual methods. The age was estimated on the basis of ring dates and information from falconers; age ranges were 4 months to 1 year for 53 falcons and >1 year for 43 falcons.
The birds were anesthetized with isoflurane delivered in oxygen, and blood samples were collected from the basilic vein with a 25-gauge needle on a 2-ml syringe. Samples were placed in 0.5-ml pediatric blood tubes that contained ethylene-diamine-tetra-acetic (EDTA) acid and were analyzed within 30 minutes of sampling. Blood smears were also prepared at the time of sampling.
Red blood cell (RBC) counts were measured by a Medonic CA620/530 VET analyzer (Boule Medical, Stockholm, Sweden) on the avian program as instructed by the manufacturer. Hemoglobin (Hg) was estimated photometrically from a whole blood sample by the HemoCue B-Hemoglobin reader (HemoCue AB, Angelholm, Sweden) with microcuvettes preloaded with reagent. Packed cell volume (PCV) was measured manually by using plain capillary microtubes and a microhematocrit centrifuge (Sigma, Osterode, Germany) at 13 845g for 5 minutes. (3) Mean corpuscular volume, mean corpuscular Hg, and mean corpuscular Hg concentration were calculated as previously described. (21)
White blood cell (WBC) counts were determined manually by dispersing 20 gl of blood into 380 gl of 1% ammonium oxalate and counting in an improved Neubauer hemocytometer (Superior Marienfeld, Lauda-Koenigshofen, Germany) under phase contrast? Blood smears were processed by the Wright-Giemsa staining procedure. (4) The WBC differential was based on 100 counted leukocytes; the differential and morphology of the cells was assessed by using an Olympus BX41 microscope (Olympus, Tokyo, Japan) under a x 100 oil immersion microscope objective. Both percentages and absolute numbers of WBC were evaluated.
Data were analyzed by Statgraphics Plus for Windows 3.0 (Statistical Graphics Corp, Herndon, VA, USA). The Kolmogorov-Smirnov test was used to determine if hematologic parameters could be adequately modeled by a normal distribution (data came from normal distribution if P [greater than or equal to] .10). Analysis of variance (for parametric data) and the Kruskal-Wallis test (for nonparametric data) were used to determine if there were significant differences between hematologic data for females, males, and birds of unknown sex. Statistical significance in all tests was set at P [less than or equal to] .05. The 95% reference interval was bounded by 2.5 and 97.5 percentiles. (22)
Results are summarized in Table 1. Percentages and absolute values of lymphocytes, monocytes, eosinophils, and basophils were not normally distributed, therefore, median values were included, and interpercentile intervals were used to calculate 95% reference intervals because the assumption of a Gaussian distribution was not required. (22) No statistically significant differences were found among females, males, and falcons of unknown sex for any of the hematologic parameters evaluated (P > .05).
This study was undertaken to determine the reference ranges of hematologic values in gyr x peregrine hybrid falcons presented for health screening at a veterinary hospital in the United Arab Emirates. Practitioners of avian and exotic medicine commonly adopt reference ranges from the literature as guidelines for interpreting hematologic results. We compared our hematologic values with those obtained by 2 other laboratories in the United Arab Emirates (Table 2). Although the number of healthy birds in our study (n = 96) was smaller than those included in reports by Wernery et al (n = 267)18 and Muller et al (n = 320), (20) it fell within the minimum number of individuals (40-100) recommended to establish reference ranges. (22)
It is apparent from the variation in values demonstrated here that clinicians should be aware of the methodology used in obtaining reference ranges before interpreting results. For example, the differences in RBC parameters, such as Hg and total RBC count, among the 3 studies can be explained by differences in methodology. Wernery et al (18) used a Cell Dyn 3500 analyzer (Abbott Laboratory, North Chicago, IL, USA) to measure RBCs, hematocrit (Hct), and estimated Hg from Hct measurements. Most of the electronic particle counters used in mammalian hematology can be used for avian RBC counts, provided that adjustments are made to compensate for larger avian cells. (1) The RBC counts reported by Wernery et al (18) were the highest among the 3 studies. In the study by Muller et al, (20) all RBC parameters were measured manually. Our laboratory used manual methods to measure PCV and Hg and the Medonic CA620/530 VET to assist with a total RBC count. The Medonic CA620/530 calibrated for falcon blood reported total RBC counts on average 16% lower than those obtained by our manual method on a selected number of paired falcon blood samples, with a correlation coefficient of 0.73 (R. Padrtova, unpublished data, March 2008). In this case, the RBC counts in our study were similar to those reported by Muller et al, (20) despite the fact that we used an automated process. The methodology we used to measure Hg was the same as that used by Muller et al, (20) and the values reported in that study were similar to those we obtained. The PCV and Hct both represent the percentage of whole blood occupied by RBCs, but these values may not be equivalent because they are determined by different methods. The machine-based Hct often differs from the manually determined PCV in that it depends on the calibration of multichannel instruments, (23,24) and PCV measurements might be artificially increased by plasma trapped among RBCs.
We recorded lower mean total WBC counts than did Wernery et al (18) and Muller et al. (20) Manual leukocyte counts differ from automated counts, and automated counts can vary among instruments. (25-27) The variation among analyzers may be the result of inherent physical differences among the analytical methods and/or calibration techniques. (24) The WBC count and differential estimated by automated hematology analyzers are not reliable in every avian species (4) because of interference by nucleated RBC and thrombocytes. (28) A previous study that used automated methods demonstrated in bustard species how a sample that contained megathrombocytes could yield a high lymphocyte count. (4) Whether the above can explain the higher automated WBC count results in gyr x peregrine falcons reported by Wernery et al (18) cannot be concluded without further investigation of WBC and thrombocyte measurements in that particular hybrid and knowledge of the calibration of the analytic instruments used. However, results of a study on chicken blood concluded that automated analysis of avian blood with the Cell Dyn 3500 and use of special avian software did not provide consistent or accurate results for certain leukocyte parameters, such as lymphocytes. (7)
Differences can also be observed when comparing differential counts. We observed higher heterophil and lower lymphocyte percentages in falcons submitted for medical assessment at our hospital compared with those reported from other laboratories. In general, absolute heterophil and lymphocyte counts are susceptible to variations influenced by stress. (13) The heterophil/lymphocyte ratios observed in untrained Harris' hawks (Parabuteo unicinctus) and peregrine falcons after the stress of transportation showed a significant increase but still remained within reference ranges for those species. (11) Stress factors, such as travel to our secluded hospital, handling, and the untrained state of many of the birds presented for initial health checks (approximately 35% of the birds), should be considered when interpreting the values. Approximately 60% of the falcons in our study were involved in active training or hunting activities, and only a few falcons were presented for health checks outside the training and hunting season. A well-designed study with birds under standardized training methods would be necessary to reveal differences in hematologic parameters among birds in training and those not being trained. In our study, assessing the levels of training was not possible, because the intensity and methods varied among falconers and clinical histories were not always reliable.
The falcon health assessment also had its limitations. The reliability of clinical signs reported depended on the experience of the falconer, which was not assessed. No virology tests or fecal or crop cultures were performed, although the quality of the mute was considered, and the crop was inspected by endoscopy.
Generally, more eosinophils and basophils were observed in our study than in those of Wernery et al (18) or Muller et al. (20) This may have been related to the differences in staining procedures. Wernery et al (18) used a slide stainer cytocentrifuge that consisted of separate eosin and thiazin (azur B, methylene blue) stains plus a light eosin rinse. Muller et al (20) applied a rapid commercially available HEMAstain. In a report on gyr falcons, May-Grunwald-Giemsa and Rapid Romanowsky stains did not adequately stain eosinophil granules, which appeared as colorless, irregular cytoplasmic vacuoles; however, the granules were appropriately stained with modified Wright-Giemsa stain. (19) We confirmed this finding in gyr x peregrine hybrids. Therefore, rather than staining smears with commercially available rapid Romanowski stains, we used a modified Wright-Giemsa stain, (4) which appropriately stained eosinophil and basophil granules. By using this stain, eosinophils appeared as large round cells with a bilobed nucleus. Purple-blue round granules of variably smaller size were scattered in a slightly basophilic cytoplasm. Basophils were smaller round cells relative to eosinophils or heterophils, and the cytoplasm contained dark purple-blue granules variable in both size and shape, which usually obscured the colorless cytoplasm and a portion or the entire round nucleus. From our experience, basophils tended to lose some of their granules when stained by rapid Romanowsky stains.
Heterophils were the granulocytes most frequently observed in our study. These appeared as large cells with a bilobed nucleus and colorless cytoplasm that contained large, cigar-shaped, pink-red granules. Lymphocytes were observed as small cells, usually round, with a dark condensed nucleus that occupies most of the pale-blue cytoplasm; occasionally, a few
azurophilic granules were also seen in the cytoplasm. Monocytes were the largest of cells, characterized by a large irregular nucleus and bluish lacy cytoplasm that sometimes contained fine eosinophilic granules. Distinguishing large lymphocytes from monocytes was facilitated by using Wright-Giemsa stain, because the monocyte cytoplasm stained clearer than with rapid stains.
Neither of the reports cited specified the sex or age of the falcons in the study groups. Obtaining the optimum number of individuals to determine if differences between sex or age exist can be challenging. Therefore, many laboratories concentrate on determining reference ranges for a species without subdividing data into subgroups defined by sex or age. We visually identified 56 gyr x peregrine falcons from our study as females, 20 as males, and 20 as unknown. We did not find statistically significant differences for any hematologic parameters among these 3 groups. In one study, leukocyte and thrombocyte counts were significantly higher in female peregrine falcons compared with males, and this finding was hypothesized to result from hormonal effects of breeding in the females, (15) Because gyr x peregrine hybrids rarely produce viable eggs and thus are not generally used for breeding, these differences were not seen in our study. (29) The birds in this study were used purely for the sport of falconry and so were not subjected to the artificially controlled stimuli required in the Middle East to bring them into breeding condition. In addition, most of the birds examined in this study were under 3 years of age and so unlikely to have come into breeding condition, especially without environmental stimuli. We were unable to accurately determine the ages of the falcons, so age was assessed from ring details, visual assessment of the bird, and information provided by the falconer. Unfortunately, both ring data and information provided by the falconer was not always reliable, and, as a result, we did not include these data. Because of the bias toward females and the statistical results, we considered the population as a whole when determining our reference ranges.
As demonstrated in our study, hematologic values of gyr x peregrine falcons obtained by different laboratories will vary. Ideally, in-house reference ranges should be used when interpreting the results of a CBC count. When adopting other reference ranges for interpretation of hematograms, the population characteristics and methodology should be taken into account.
Acknowledgments: We thank Mrs Ruchira Sepali and Mr Shaiju Abbas for the technical assistance in the laboratory, and Dr Mirjam Hampel, Dr Tim Shaw, and Dr Giulio Russo for their assistance in health assessment of the falcons.
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Renata Padrtova, MSc, PhD, and Christopher G. Lloyd, BVSc, MSc, CertZooMed, MRCVS
From the Nad Al Shiba Veterinary Hospital, PO Box 116345, Dubai, United Arab Emirates.
Table 1. Hematologic values of clinically normal gyr X peregrine hybrid falcons (N = 96). (a) Parameter Mean SD Red blood cells (X [10.sup.12]/L) 2.68 0.23 [X [10.sup.6]/[micro]l] Hemoglobin (g/L) 179.20 10.90 [g/dl] [17.9] [1.1] Packed cell volume (%) 47 3 Mean corpuscular volume (fl) 176.50 10.30 Mean corpuscular hemoglobin (pg) 67.30 5.10 Mean corpuscular hemoglobin concentration (g/L) 381.3 20.4 [g/dl] [38.1] [2.0] White blood cells (X [10.sup.9]/1-) 5.63 1.63 [X [10.sup.3]/[micro]l] Heterophils X [10.sup.9]/L 3.98 (69.9) 1.41 (10) [X [10.sup.3]/[micro]l] (%) Lymphocytes X [10.sup.9]/L 1.39 (25.3) 0.54 (9.2) [X [10.sup.3]/[micro]l] (%) Monocytes X [10.sup.9]/L 0.10 (l.8) 0.09 (1.6) [X [10.sup.3]/[micro]l] (%) Eosinophils X [10.sup.9]/L 0.10 (1.9) 0.11 (1.9) [X [10.sup.3]/[micro]l] (%) Basophils X [10.sup.9]/L 0.03 (1.1) 0.03 (1.2) [X [10.sup.3]/[micro]l] (%) Parameter Median Minimum Red blood cells (X [10.sup.12]/L) 2.66 2.19 [X [10.sup.6]/[micro]l] Hemoglobin (g/L) 187.00 152.00 [g/dl] [18.7] [15.2] Packed cell volume (%) 47 39 Mean corpuscular volume (fl) 177.70 153.20 Mean corpuscular hemoglobin (pg) 67.40 56.20 Mean corpuscular hemoglobin concentration (g/L) 380.4 338.7 [g/dl] [38.0] [33.9] White blood cells (X [10.sup.9]/1-) 5.45 2.15 [X [10.sup.3]/[micro]l] Heterophils X [10.sup.9]/L 3.82 (71.5) 1.46 (43.0) [X [10.sup.3]/[micro]l] (%) Lymphocytes X [10.sup.9]/L 1.29 (23.0) 0.47 (9.0) [X [10.sup.3]/[micro]l] (%) Monocytes X [10.sup.9]/L 0.08 (2.0) 0 (0) [X [10.sup.3]/[micro]l] (%) Eosinophils X [10.sup.9]/L 0.07 (1.0) 0 (0) [X [10.sup.3]/[micro]l] (%) Basophils X [10.sup.9]/L 0.03 (1.0) 0 (0) [X [10.sup.3]/[micro]l] (%) Parameter Maximum 95% Reference interval Red blood cells (X [10.sup.12]/L) 3.33 2.28-3.29 [X [10.sup.6]/[micro]l] Hemoglobin (g/L) 204.00 157.0-201.0 [g/dl] [20.4] [15.7-20.1] Packed cell volume (%) 54 40-52 Mean corpuscular volume (fl) 203.50 157.7-194.7 Mean corpuscular hemoglobin (pg) 83.60 59.1-78.2 Mean corpuscular hemoglobin concentration (g/L) 459.0 3.44.0-441.5 [g/dl] [45.9] [34.444.2] White blood cells (X [10.sup.9]/1-) 9.65 2.95-9.05 [X [10.sup.3]/[micro]l] Heterophils X [10.sup.9]/L 7.51 (85.0) 1.68-6.99 (45.0-84.0) [X [10.sup.3]/[micro]l] (%) Lymphocytes X [10.sup.9]/L 2.71 (53.0) 0.64-2.35 (13.0-48.0) [X [10.sup.3]/[micro]l] (%) Monocytes X [10.sup.9]/L 0.47 (8.0) 0-0.32 (0-6.0) [X [10.sup.3]/[micro]l] (%) Eosinophils X [10.sup.9]/L 0.56 (8.0) 0-0.40 (0-6.0) [X [10.sup.3]/[micro]l] (%) Basophils X [10.sup.9]/L 0.14 (5.0) 0-0.11 (0-4.0) [X [10.sup.3]/[micro]l] (%) The values are in SI units; conventional units are in brackets. Table 2. Comparison of published hematologic values (mean [SD]) of gyr X peregrine hybrid falcons (a) Present study Wernery et al (18) Parameter (no. falcons = 96) (no. falcons = 267) Red blood cells 2.68 [+ or -] 0.23 3.35 [+ or -] 0.12 (X [10.sup.12]/L) [X [10.sup.6/[micro]l] Hemoglobin (g/L) 179.2 [+ or -] 10.9 153.3 [+ or -] 6.2 [g/dl] [17.9 [+ or -] 1.11 [15.3 [+ or -] 0.61 Packed cell volume (%) 47 [+ or -] 3 -- Hematocrit (%) -- 46 [+ or -] 2 Mean corpuscular volume (fl) 176.5 [+ or -] 10.3 137.3 [+ or -] 4.2 Mean corpuscular 67.3 [+ or -] 5.1 45.7 [+ or -] 3.4 hemoglobin (pg) Mean corpuscular hemoglobin concentration (g/L) 381.3 [+ or -] 20.4 no data [g/dl] 38.1 [+ or -] 2.0 no data White blood cells 5.63 [+ or -] 1.63 9.31 [+ or -] 3.24 (X [10.sup.9]/L) [X [10.sup.3]/[micro]l] Heterophils (%) 69.9 [+ or -] 10.0 60.0 [+ or -] 10.0 Lymphocytes (%) 25.3 [+ or -] 9.2 37.4 [+ or -] 11.2 Monocytes (%) 1.8 [+ or -] 1.6 2.6 [+ or -] 1.2 Eosinophils (%) 1.9 [+ or -] - 1.9 0 Basophils (%) 1.1 [+ or -] 1.2 0 Muller et al (20) Parameter (no. falcons = 320) Red blood cells 2.39 [+ or -] 0.26 (X [10.sup.12]/L) [X [10.sup.6/[micro]l] Hemoglobin (g/L) 179.0 [+ or -] 15.7 [g/dl] [17.9 [+ or -] 1.61 Packed cell volume (%) 52 [+ or -] 4 Hematocrit (%) - Mean corpuscular volume (fl) 219.7 [+ or -] 25.4 Mean corpuscular 75.6 [+ or -] 8.5 hemoglobin (pg) Mean corpuscular hemoglobin concentration (g/L) 344.3 [+ or -] 14.8 [g/dl] 34.4 [+ or -] 1.5 White blood cells 7.55 [+ or -] 2.27 (X [10.sup.9]/L) [X [10.sup.3]/[micro]l] Heterophils (%) 49.9 [+ or -] 3.5 Lymphocytes (%) 44.2 [+ or -] 3.4 Monocytes (%) 4.4 [+ or -] 1.6 Eosinophils (%) 1.4 [+ or -] 1.1 Basophils (%) 0.1 [+ or -] 0 (a) The values are in SI units; conventional units are in brackets.
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