(D) Data points represent absolute WBC count for individual female mice (N=10, 2 separate experiments) and bars represent mean value for each time point. * Significantly different compared to cardiac. Values represent mean ± SEM for (B) female or (C) male mice (N=10, 2 separate experiments). (A) Representative flow plots showing gating strategy used to define blood cell populations. No significant difference was found in CBC parameters, indicating that alterations are the result of site and manner of sampling. To evaluate whether CBC differences noted for facial, cold tail and warm tail reflected bleeding method or were related to absence of anesthesia (cardiac and retro-orbital were performed under isoflurane), additional mice were bled by either facial, cold tail, or warm tail under isoflurane ( Supplemental Figure 1). In contrast to WBC parameters, total red blood cells and platelets, hematocrit, and hemoglobin concentration, were equivalent for cardiac and retro-orbital, but increased for facial and cold and warm tail. The percentages of different cell types were less affected by bleeding method and no difference was noted between tail bleeding methods. Both methods of tail bleeding produced significantly higher WBC, neutrophils, and lymphocytes, but warming significantly reduced these increases compared to cold tail. Facial bleeding yielded results comparable to cardiac. Using central cardiac bleeding as a baseline, comparison of the methods showed that retro-orbital bleeding consistently resulted in the lowest absolute number of white blood cells (WBC), neutrophils, lymphocytes, and monocytes in both male and female mice ( Figure 1). To assess the effect of bleeding method on normal peripheral blood cell values, mice were bled by five different methods as described in the methods section. We analyzed hematologic parameters using five common blood sampling sites/methods and compared differences in blood cell populations and function to assess the impact of bleeding method on acquired data. To our knowledge, the effects of warming or sampling method on phenotype-defined blood cells or the effects of bleeding method on blood cell progenitors have not been evaluated. However, they did not comprehensively evaluate collection sites nor utilize current anesthetics, automated cell counters or flow cytometry. Classic studies have reported differences in CBC and chemistry values from limited sampling sites in rodents 2 4 7– 13. Common sites for blood collection in mice include the retro-orbital sinus, facial vein (superficial temporal or submandibular veins), tail vein, saphenous vein, and heart (cardiac puncture) 2 4– 6. Age, gender, and strain can affect complete blood count (CBC) and blood chemistry values 1– 3 and is the basis for including animal source and strain in study designs. Identifying ways to decrease variability in measured parameters and standardize comparison amongst laboratories are important in order to minimize animal usage and improve data evaluation. Our findings suggest that the same method should be used across an entire study, that warming of mice prior to tail bleeds should be done to uniform levels, and that accurate description of bleeding methods in publications should be provide to allow for interpretation of comparative reports and inter- and intra-laboratory experimental variability. Intriguingly, warming of mice prior to tail bleeding was shown to significantly alter blood values. In this study, we report significant alterations in blood parameters, particularly of total white blood cells, specific populations of dendritic cells and myeloid derived suppressor cells, and hematopoietic progenitor cells as a result of site and manner of sampling. The method of blood acquisition varies considerably across laboratories and is often not reported in detail. Commonly used sites in the mouse include the retro-orbital sinus, facial vein, tail vein, saphenous vein, and heart. However, the small body size of the mouse limits collection based upon volume, frequency, and accessible sites. Often, analysis of blood occurs in a non-terminal manner. The laboratory mouse is the most frequently used animal model for hematologic studies and for assessment of blood parameters across a broad range of disciplines.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |