|Nejra Hadzimusic1*, Muhamed Katica2, Zijad Muharemovic3, Jasmin Musanovic4
|Corresponding Author: Nejra Hadzimusic, Department of Pathophysiology, Veterinary Faculty, University of Sarajevo, Bosnia and Herzegovina; Email: [email protected]|
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Background: Hematology results are often influenced by the time between blood sampling and measurement, as well as storage conditions (e.g., temperature and time) during sample delivery between laboratories may further affect the resulting data. Hematological changes may occur in the measured parameters as a consequence of delayed analysis and may complicate interpretation of the data. Delayed analysis of blood samples may be caused by restricted access to laboratories. Blood samples collected from turkeys in remote locations (farms) often wait for laboratory processing.
Objectives: The aim of this study was to investigate changes that occur in the packed cell volume (PCV), hemoglobin concentration, red blood cell (RBC) counts and mean corpuscular values (MCV) of turkey blood samples due to storage, including: blood samples stored at refrigerator (4°C), laboratory (24°C) and water bath (33°C) temperatures across a storage period of 72 h.
Methods: Blood samples were collected from 25 adult turkeys (British United Turkey 600 hybrids – BUT 600). Hematological determinations were carried out on the blood samples immediately upon collection to obtain the baseline value (BV) and thereafter at specific time intervals across the 72-h duration of storage (DOS).
Results: Results showed that for the samples stored at 4°C, there were no significant changes at p<0.05 level from the BV in the PCV, hemoglobin concentration and RBC counts, all through the 72-h DOS, but significantly increased values (p<0.05) were obtained when samples stored at 24°C and 33°C.
Conclusion: Based on our research results we concluded following: blood samples obtained from turkey stored up to 72 hours at 4°C provides legitimate results for PCV, Hgb concentration and RBC count. MCV value is reliable if blood sample was stored up to 30 hours.
|Turkey blood, Hematology, Duration of storage, Storage temperature|
|Avian clinical hematology has become a vital part of a series of routine laboratory tests that aid veterinary clinicians to arrive at a diagnosis, make a prognosis and/ or assess the efficacy of therapeutic interventions in avian clinical practice1, 2, 3. Because avian blood does not store well (e.g. during transport) hematologic results obtain soon after collection are preferred over those preferred several hours later2, 4, 5. Although it is recommended to perform laboratory analysis immediately upon collection, often it is not possible, especially when blood samples are collected from remotely located farms. Unfortunately, due to often used manual procedure (instead of automated hematology analyzers), blood samples may not be analyzed immediately after arrival at the laboratory. Automated hematology instruments are used for mammalian blood analysis, but there is a lack of accurate automated methods available for avian blood analysis. Manual procedures are commonly used for the determination in avian hematology, because all blood cells are nucleated1, 2, 3. It is well known that handling of blood samples, as well as method of keeping and storage can significantly influence the results of hematological determinations. Consequently, results of hematological determinations of improperly stored or handled blood samples can yield misleading results6, 7, 8, 9, 10. Butarrello10 and Goosens11 in their studies concluded that refrigeration of human blood samples is recommended to stabilize blood and minimize artifactual changes. Research conducted on different animal species showed significant differences in the stability of blood samples stored at room or refrigerator temperatures12, 13, 14. Study conducted by Bluel12 on bovine blood showed that refrigeration had a stabilizing effect on red blood cells count, but led to decrease in white blood cells count during 24 hours of storage. Studies on equine blood conducted by Clarke15 showed that hematological parameters were more stable in blood samples kept at room temperature than those kept in refrigerator.|
|So far, according to authors’ knowledge, there had been no reports on the effects of temperature and duration of storage period on the hematological parameters in turkey blood sample. Therefore, this research has been conducted in order to detect changes in the hematological values of turkey blood samples, stored for up to 72 hours at refrigerator (4°C), room (24°C) and water bath (33°C).|
Material and methods
|Blood samples were collected from 25 turkeys (British United Turkey 600 hybrids – BUT 600). Animals were procured as 1-month old from turkey producer (Jasmin d.o.o., Orašje, Bosnia and Herzegovina). Turkeys were raised in extensive production. A total of 25 blood samples were collected from apparently healthy adult turkeys. Blood samples were collected by ulnar vein-puncture method in tubes á 15 ml with anticoagulant. The blood samples were collected in the morning hours. Immediately upon collection hematological determinations were carried out on the blood samples, to obtain the baseline value (BV). Afterwards, the blood sample from each turkey was gently mixed and shared into three equal parts. One part was kept in refrigerator (temperature 4°C), another was kept at room temperature (24°C) while third part was kept in a water bath (temperature 33°C). Hematological determinations were carried out during 72 hours of storage on different temperatures, as follows: firstly, three hour intervals during first 12 hours of storage (hour 3, 6, 9 and 12), then at six-hour intervals during next 24 hours (hours 18, 24, 30 and 36) and at twelve-hour intervals for the remaining 36 hours (hours 48, 60 and 72).|
|The blood samples were used for hematological parameters including red blood cells (RBC) count, packed cell volume (PCV), hemoglobin (Hgb) concentration, and mean corpuscular volume (MCV). RBC count was determined by using Neubauer hemocytometer (Assistant, Germany). PCV was determined by using microhematocrit method6 using microhematocrit centrifuge and reader (Hawksley, England), expressed in percents (%). The hemoglobin concentration was determined by using hemoglobinometer (Hawksley, England) and expressed in conventional Units g/dL (Conversion factor to SI Units: x10). MCV was calculated using the standard formula3.|
|Results of the study were presented as mean values with standard errors (SE) of each parameter determined at specific time intervals starting from the BV. Data was analyzed for statistical differences using ANOVA and the result of each determination was compared with BV.|
|Mean PCV value in blood samples stored at different temperatures (4°C, 24°C and 33°C) from BV (33,0±0,58%) determined immediately after collection increased to maximum value in hour 72 (36,7±0,54%) in samples stored at 4°C; 43,12±2,5% in samples stored at 24°C and 55,0±1,9% in samples held at 33°C (Fig. 1.). There were no significant differences at p<0.05 between the BV and values determined in samples stored at 4°C during period of 72h (Table 1.). Statistically significant differences (p<0.05) were noticed between BV and 24h from collection in samples stored at 24°C and 33°C (Tab. 1).|
|for long because of storage-related degenerative changes that occur in the RBCs that lead to widening of the “pores” on the surface of the RBCs, which permit ingress of water into the cells. Increases in PCV and MCV like those mentioned above were reported also in horses15, bovine12, 14 pigs, goats and rats14 and chicken20.|
|In our study, storage temperature of 4°C significantly decreased changes regards to PCV, as well as MCV. Higher temperatures (24°C and 33°C) yet caused greater RBC swelling. Our findings are consistent with studies conducted on human blood samples7 as well as cattle12, 14, goats, pigs14 and rats13, but in contrast with the study conducted by Clarke15 on equine blood samples, which showed artifactual changes in stored blood samples. Study showed increased numbers of macrocytic hypochromic RBCs. Changes were, according to Clarke15 less pronounced in samples stored at 24°C than at 4°C.|
|Significant decrease of hemoglobin concentrations and RBC counts in blood samples stored at 33°C from BV was noticed after 60 hours from blood collection might be outcome of higher temperature induced conversion of some of the hemoglobin intermediates and due to significant autolysis of the RBCs associated with higher temperatures of storage8, 21.|
|Based on the results, we concluded that the blood samples obtained from turkeys stored up to 72 hours at 4°C provide legitimate results for PCV, Hgb concentration and RBC count. MCV value is reliable if blood sample was stored up to 30 hours. Blood samples stored at 24 °C can be used for hemoglobin concentration and RBC count up to 72 hours of storage, but for PCV only up to 18 hours and MCV up to 12 hours from sample collection. Samples kept at 33°C give reliable results for MCV up to 12 hours, PCV up to 18 hours and hemoglobin concentration and RBC count up to 48 hours from blood collection.|
|Results of our research can be used as a guide to determine the appropriate storage and handling of turkey blood samples. We also recommend further research based on shorter intervals during 72 hours of storage as well as further general discussion of sample collection and handling, and discussion on considering some of the routinely encountered problems (and how to avoid them) associated with performing commonly requested tests (e.g. complete blood cell counts, chemistry profiles, etc.).|
|List of abbreviations:|
|BUT 600 - British United Turkey 600 hybrid|
|BV – baseline value|
|PCV - packed cell volume|
|RBC - red blood cell|
|Hgb – hemoglobin|
|MCV - mean corpuscular value|