In this study, significant differences were observed between the total peripheral leukocyte counts of blood samples collected via the cardiac puncture and the other three methods applied in mice; tail vein, foot vein, and saphenous vein punctures (Figure ). It was consistently observed that leukocyte counts were lowest in blood collected from the heart. The blood samples collected by cardiac puncture had leukocyte counts of about 2–3 folds less than those obtained by use of the other techniques, i.e., tail, foot, and saphenous. There were no significant differences in total leukocyte counts among the samples taken from the tail, foot, and saphenous veins as compared to each other. In addition, of the four methods applied, cardiac puncture appeared to be the fastest technique, yielding the maximum blood volume with little physical stress being applied to the site of blood sampling. A fairly large volume of blood (i.e., 0.7 to 1 ml) can easily be collected via a cardiac puncture, while a much smaller volume (30 to 100 ul) is possible to be collect through the tail, foot, or saphenous vein punctures with variable degrees of difficulty. However, an advantage of blood drawing from the foot, saphenous, or tail is that it can be performed on a conscious mouse if application of a light anesthetic is not an option. On the other hand, cardiac puncture requires the use of anesthetics.
Figure 1 Peripheral blood leukocyte counts in male and female mice. Blood samples were collected into heparinized containers from male and female mice via cardiac, tail vein, foot vein, and saphenous vein punctures. Data are expressed as mean ± SEM (x10 (more ...)
Experimental animals with different genders are applied in various studies and gender dimorphism appears to play a role in host response. Thus, it was of interest to examine the effect of sampling sites on the level of blood leukocytes found in female mice. Because there were no significant differences between blood samples collected from the tail, foot and saphenous veins, only two of these techniques, i.e. tail and saphenous, were applied along with cardiac puncture in this part of the study. Similarly, the results indicated that blood collected from the female heart had the lowest leukocyte counts when compared to blood collected from the tail and saphenous veins (Figure ). While there were no significant differences between the total leukocyte counts of male and female blood samples, a significant difference in differential leukocyte counts was observed between female and male blood samples obtained by cardiac puncture. The percentage of neutrophils in blood collected via cardiac puncture in female mice was significantly lower than those in male mice (Table ). Additionally, when the absolute numbers of neutrophils were calculated the difference was statistically significant between the neutrophil counts of blood collected from the hearts of male and female mice (absolute neutrophil counts: female: 0.196 ± 0.05 × 106
/ml, and male: 0.638 ± 0.136 × 106
< 0.001). There were no significant differences between blood samples of male and female mice obtained from the tail vein or saphenous vein. The data suggests that sex steroids have an effect on the regulation of circulating leukocytes. It has been shown that female steroid hormones, 17 beta-estradiol and progesterone, can modulate the expression of adhesion molecules on human peripheral blood leukocytes, thereby, affecting their circulation and activation [11
]. In women, a consistent fall in neutrophil count at menstruation has been shown [12
]. The fall in neutrophil counts may be related to infiltration of neutrophils into the uterus as has been demonstrated in mice treated with estradiol [13
]. Further, the extensive experience of our laboratory (un-published data) of working with human blood leukocytes as well as published studies [14
], indicate a great variation in the concentrations and cellular responses of white blood cells of healthy adult female individuals, which correlate to various phases of the menstrual cycle. Other published studies in animals have also indicated the influence of the reproductive state (i.e., estrous cycle) on neutrophil concentrations and functions [10
]. It must be noted that in our study presented here, the phases of the estrus cycle were not identified. Further study is required to evaluate the role of sex hormones and cyclic changes on the concentrations of peripheral blood cells. The data presented in this study and the previously published studies collectively suggest the effect of sex steroids on circulating leukocytes, which should be considered in experimental design and interpretation of data.
Table 1 Peripheral blood leukocyte differential counts in male and female mice. Blood samples were collected into heparinized containers from male and female mice via cardiac, tail, and saphenous punctures. Blood smears were prepared and stained with the Wright (more ...)
Despite the significant differences presented between the neutrophil counts of blood samples drawn from the hearts of female and male mice, no significant difference was present when blood samples were drawn from tail and saphenous veins. One potential explanation may be due to the influence of the sampling techniques. In blood samples taken from the tail and saphenous, local physical stress may have caused the release of marginating leukocytes into circulation, falsely changing the leukocyte concentrations of the circulating blood. However, such local manipulation was not introduced during the cardiac puncture. Therefore, the effect of female steroid hormones on circulating leukocytes can easily be appreciated in blood drawn from the heart and not of the samples collected from the tail and saphenous veins. Some investigators have speculated that differences in vessel diameter and flow dynamics account for the leukocyte counts obtained at various sampling sites. No striking variation in vessel diameter was observed between the male and female mice used in our study. Although the existence of flow dynamic variability between male and female mice was not examined in this study, the male and female mice appeared to exhibit similar heart beats. However, the variability in vessel diameter could be a factor contributing to the differences that existed between blood leukocyte counts collected from the heart, tail, foot, and saphenous.
The results of our study of mice are in accord with the results of similar studies involving other rodents [6
]. A marked decrease in leukocyte counts has been reported in samples taken from the heart versus the tail of rats [4
]. Additionally, Nemzek et al. have shown significantly lower leukocyte counts in blood collected from the mouse heart when compared to those of the tail and orbital sinus [7
]. However, the leukocyte counts of the mice blood in our study (tail: 8.4 ± 0.9 × 106
/ml, heart: 2.7 ± 0.5 × 106
/ml) were lower than those of Nemzek et al. (tail: 16 ± 2 × 106
/ml, heart: 4 ± 1 × 106
/ml). The differences may due to the differences in sampling techniques. Nemzek et al. clipped the tail and did a laparotomy to expose the heart for blood sampling. In our study, the tail vein was punctured with a 25-guage needle and blood was collected into a heparin-coated capillary tube. Further, blood was drawn from the heart without the animal being subjected to any surgical procedure for laparotomy. In the Nemzek et al. study, the trauma induced by clipping the tail and the surgical laparotomy might have triggered signals to release the marginated leukocytes into circulation, and thereby reflecting the higher number of leukocyte counts. Another reason may due to the procedure used in blood analysis, in which Nemzek et al used an automated hematology system cell counter while in our study the cell counts were performed manually using a hemocytometer. Furthermore, different anesthetics were used in our study (i.e., methoxyflurane) than those of Nemzek et al. (i.e., ketamine, and xylazine). Studies have shown that the type of anesthetics may have an effect on leukocyte counts [4
]. However, the data presented in this study as well as our un-published data from un-anesthetized mice have shown no significant effect of the anesthesia on leukocyte counts of blood collected from the tail, foot and saphenous veins. We have not drawn any blood from the hearts of un-anesthetized mice. Therefore, one must note that the relative counts may change if different anesthetics are applied. Our study supports the previous reports and adds new information regarding blood parameters collected via the saphenous vein and cardiac puncture without opening the animal.
The reason(s) for the differences observed in the leukocyte counts collected from different sampling sites is not clearly understood. One reason may be attributed to the immunological demands of the host at various sites [14
]. Another theory is that the vascular resistance and blood stasis created by the larger surface area in the capillary bed may be responsible for the higher leukocyte counts from peripheral sites [5
]. Further, it is possible that stress-induced procedures at blood sampling sites may cause changes in the number of leukocytes. For example, there is a great deal of manipulation involved in locating the saphenous, foot, and tail veins for drawing. The areas are rubbed and massaged, while pressure is applied in order to find a good visible vein beneath the skin. On the other hand, this type of manipulation is absent in the cardiac puncture method. In fact, in our experience, the cardiac technique was the easiest of all the techniques to perform. The puncture was made quickly and efficiently, and the blood flowed easily from the beating heart into the syringe. However, further manipulation was often needed after a puncture was made in the other techniques to prevent coagulation and facilitate further flow of blood. Drawing from the saphenous vein was the next best sampling site when compared to the tail and foot veins. This may be attributed to the larger size of the blood vessel, which facilitated a better blood flow with less physical manipulation applied to the site. Additionally, physical manipulation induced by rubbing and/or local warming by exposing the animal to 37°C for a few minutes, causes vasodilatation, which promote bleeding [16
]. Further, mechanical stimulation induced by rubbing the area causes local release of mediators resulting in the release of leukocytes from the marginal pool into the blood stream, effecting the increase in leukocyte counts. One might argue that the removal of blood via multiple sampling of the tail, foot, and saphenous veins have affected the leukocyte counts of the blood drawn from the heart at the end of the blood sampling. We don't believe this is a significant factor in our studies for the following reasons. We have observed no significant differences between leukocyte counts of blood collected from mice that have only been subjected to cardiac puncture (data not shown in this report) and those that have been subjected to multiple blood draws as shown in this study. Additionally, a study has shown that changing the order of blood sampling did not alter the results of leukocyte counts in mice [7
]. Further, studies have shown that removal of blood beyond 15% can affect some of the hematological parameters such as mean corpuscular volume and red cell counts [16
]. In our study, a total of 120 ul of blood were drawn via tail, foot, and saphenous vein punctures, which is less than 10% of total blood volume of the mouse (a mouse of 25 g body weight has about 1.8 ml blood volume) [16
In summary, while it is recognized that the cardiac puncture technique is not always practical and cannot be used in ongoing studies, one should at least be aware of the differences in blood variables as a result of sampling technique and gender dimorphisms. Accordingly, investigators should choose the gender and method of blood draw that best fits their needs regarding anesthetic availability, sample volume, and frequency of sample collection, to be used consistently throughout the study. In addition, one should take these differences into account when comparing studies that have employed varying methods of blood collection and genders.