Comparison of a New Blood Sampling Device With the Vacuum Tube System for Plasma and Hematological Analyses in Healthy Dogs

Introduction

Blood sampling is routinely performed in small animals. The current reference technique involves direct sampling from the jugular vein in evacuated tubes.¹ This technique has several drawbacks, however. The vacuum effect may increase blood extravasation as the needle is removed. Also, in the authors’ experience, the risk of hematoma exists, especially when blood is repeatedly sampled from the same vein. To avoid this risk, one should wait until the tube is filled with blood before removing the needle. The minimal volume of blood collected is 2 or 3 mL; this amount conforms to the ratio of blood to anticoagulant volume that is required and may be of importance in very small patients, especially when repeated biochemical or hematological examinations are performed over a limited period of time.¹

The vacuum may be excessive in small animals (e.g., miniature dogs or puppies) or those with poor circulation. This sometimes leads to collapse of the vein and the inability to collect the required blood volume.² Moreover, when venipuncture is not direct, contamination of the specimen with enzymes or substrates from tissue surrounding the vein may occur when vacuum is applied.³

The anatomical location of the vein means that the head of the animal must be held in a fixed position, which may be uncomfortable and stressful for the animal. In addition, sudden movement once the needle has been inserted may theoretically prove hazardous due to the close proximity of the carotid artery and trachea.

For all the above-mentioned reasons, practitioners frequently use alternatives to vacuum systems. Blood may be sampled from the jugular vein with syringe and needle to limit an excessive vacuum and to better control the volume collected. Many practitioners also draw blood with a syringe and needle, but they collect from the cephalic vein instead, because the position required for this procedure is generally better tolerated by dogs. Nevertheless, as for any blood sampling technique, the risk of vein collapse and hematoma may persist, and the blood may coagulate if the sampling time is too long and mixing of the blood with the anticoagulant is delayed.

Use of pediatric sampling devices based on a capillary system overcomes several previously mentioned drawbacks. In particular, no vacuum is applied and a very small volume of blood is taken. This technique can be used at the cephalic vein, which implies a more comfortable position for the animal. A 25-gauge needle (i.e., about 30% smaller than the conventional 22-gauge needle traditionally recommended with vacuum tubes) can be used for venous puncture.

The aim of this study was to compare use of a sampling device,^a recently shown to be reliable in humans,⁴ with the current so-called reference technique by measuring routine hematological and biochemical parameters in healthy dogs. Each dog underwent each blood sampling technique consecutively, once in the morning and once in the afternoon, with the sequence inverted to allow comparison within the same individuals. Finally, a plasma exogenous creatinine clearance test was done to compare the two techniques for repeated sampling in a dynamic functional assay.⁵

Materials and Methods

This study was conducted in November 2005 according to conditions approved by the French Ministry of Agriculture and according to the guidelines of the Guide for Care and Use of Laboratory Animals.^b

Seven intact beagles (five females, two males), aged between 2 and 17 years and weighing 9.5 kg to 16.9 kg, were used. The dogs were considered healthy according to their medical records, physical examinations, and previous plasma and urine panels. Each dog was sampled four times on the same day by the same operator, who was well trained in both sampling methods. The same assistant restrained each dog in a sitting position.

In the morning, dogs were sampled first from the right jugular vein with vacuum tubes and 5 minutes later from the right cephalic vein with capillary systems. Inversely, in the afternoon, dogs were sampled in the same order—first from the right cephalic vein with capillary systems and 5 minutes later from the right jugular vein with vacuum tubes. The samples were stored at 4¢ªC and assayed within 30 minutes. Two tubes of blood (an ethylenediaminetetraacetic acid [EDTA] tube for hematological analyses and a lithium heparin tube for plasma biochemical analyses) were taken each time from the jugular vein (with a 22-gauge needle and vacuum tubes;^c 3 mL of blood per tube) and from the cephalic vein (with a 25-gauge needle and capillary tubes;^d 0.2 mL of blood per tube). For blood sampling with the capillary system, the needle was inserted in the cephalic vein, and a tube was placed in position when a drop of blood appeared through the needle hub, so the blood then filled the tube [Figures 1, 2]. For each technique used, the times taken to complete the entire sampling procedure (i.e., from the start of restraint to completion of the sample procedure: time A) and to fill the two tubes once the needle was inserted (time B) were recorded.

Plasma biochemical assays were performed with an analyzer. ^e The heparinized tubes were centrifuged (10 minutes, 3000 g), and plasma (about 100 μL for the capillary system) was collected. Five parameters were measured: glucose, creatinine, alanine aminotransferase (ALT), alkaline phosphatase (ALP), and potassium (K). Complete blood counts including white blood cell count (WBC), estimated counts of granulocytes (eGC), eosinophils (eEC), monocytes (eMC), lymphocytes (eLC), red blood cell count (RBC), hemoglobin concentration (Hb), hematocrit (Ht), mean corpuscular volume of erythrocytes (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), mean platelet volume (MPV), and red cells distribution width (RDW) were performed with an analyzer.^f A blood smear was made from the specimen for manual determination of the differential count of leukocytes (neutrophils [NC], eosinophils [EC], basophils [BC], lymphocytes [LC], and monocytes [MC]), and packed cell volume (PCV) was also manually measured using microhematocrit tubes.

The plasma exogenous creatinine clearance test was performed, as previously described, on 2 consecutive days in six dogs.⁵ The test substance was aqueous creatinine solution (80 mg/mL) prepared with anhydrous creatinine^g and sterile distilled water. Creatinine was administered as an intravenous bolus at a dosage of 40 mg/kg. Blood samples were taken just before the administration and at 5, 10, and 30 minutes and 1, 2, 4, 6, and 8 hours after the administration. Case nos. 1, 2, and 3 were sampled from a jugular vein with the vacuum tube system, and case nos. 4, 5, and 6 were sampled from a cephalic vein with the capillary system on day 1 and conversely on day 2. Samples were taken in heparinized tubes and stored at 4¢ªC; then they were assayed within 30minutes. Plasma creatinine was measured with an analyzer^e after centrifugation (10 minutes, 3000 g). Glomerular filtration rate was determined from the plasma clearance of exogenous creatinine, calculated with the appropriate software.^h

Statistical analyses were performed with a statistical software package.ⁱ Results are expressed as mean ± standard deviation (SD). Hematological, biochemical, and glomerular filtration rate values obtained from cephalic veins with capillary systems (M) were compared to those obtained from jugular veins with vacuum tubes (V) according to the following general linear model:⁶

with:

Y_i,j,k being the value observed for dog i with technique j in period k;

μ being the general mean effect;

Dog_i being the effect of dog i (i = 1 to 7);

Technique_j being the effect of the sampling technique (j =M or V);

Period_k being the effect of time of sampling (k = morning or afternoon); and

ϵ_i,j,k being the error of the model.

A P value <0.05 was considered significant.

For hematological and biochemical values, the difference plot method as described by Bland and Altman was used to illustrate the difference in test results between the two techniques: the x axis shows the mean of the results of the two methods ([M + V]/2), whereas the y axis represents the absolute difference between the two methods ([M - V]).⁷

Results

Both techniques were well tolerated by the dogs, and no complications related to sampling procedures (e.g., failure or repeated sampling attempts, vein collapse, injury, hematoma) were observed with either technique. Twenty-eight samples were obtained for the biochemical and hematological assays. The sampling technique did not affect time A (1.4±0.95 and 1±0.60 minutes, respectively, for M and V) or time B (1.2±0.84 and 0.6±0.59 minutes, respectively, for M and V).

The mean ± SD and range for each variable tested are given in Table 1 for M and V. Table 2 provides values for the maximal differences observed between M and V, the median of the differences, and the reference intervals of the analytical methods used. No period effects (i.e., morning versus afternoon sampling) were observed except for ALP and eEC. The morning and afternoon values were 135±43 and 131±40 U/L, respectively, for ALP (P=0.033) and 0.21±0.07 and 0.16±0.06 10³/μL, respectively, for eEC (P=0.003). No difference was observed between the two techniques for creatinine, ALP, and K.

Glucose and ALT were the only biochemical variables for which the values differed significantly according to the sampling technique. The largest differences observed between M and V were 0.6 mmol/L for glucose and 26 U/L for ALT, respectively. The techniques were compared by the Bland-Altman method [Figures 3, 4], which showed that M results, compared to V, overestimated plasma glucose and ALT.

Six of 14 hematological parameters assessed by the analyzer (WBC, eLC, eGC, RBC, Hb, and Ht) showed statistically significant differences according to the blood sampling technique. The largest differences observed between M and V were 1.5 10³/μL for WBC, 0.3 10³/μL for eLC, 1.1 10³/μL for eGC, 0.62 10⁶/μL for RBC, 1.6 g/dL for Hb, and 4.1% for Ht, respectively. With manual differentials, the sampling technique significantly affected NC and EC, with a maximal observed difference of 1.37 10³/μL and 0.39 10³/μL, respectively. The PCV in microhematocrit tubes also differed, with a maximum difference between the two techniques of 6% in one dog. The techniques were compared for all of these hematological parameters by the Bland-Altman method [Figures 5 through 13], which showed that WBC, eLC, eGC, RBC, Hb, Ht, and NC were overestimated with the M technique compared to the V technique, whereas EC and PCV were underestimated.

A total of 108 samples were taken for glomerular filtration rate assessment. The two curves for each dog over-lapped [Figure 14]. The observed values for plasma clearance of exogenous creatinine were 3.8±0.71 and 3.7±0.61 mL/kg per minute for M and V, respectively. No statistically significant effect of M and V was observed.

Discussion

The main finding of this study is that the M technique may offer an appropriate alternative to the V technique for both hematological and plasma analyses in healthy dogs. In healthy humans, skin puncture using the M technique was shown to provide very similar results to those obtained with venipuncture using the V technique.⁴ The effects of sampling procedures on plasma variables are poorly documented. Only a few results exist for the effect of the site of sampling. Some plasma variables, like plasma lactate, creatinine, and K concentrations were indeed shown in other studies to depend significantly on sampling site in dogs.^8,9 Some authors have developed specific sampling techniques for monitoring glucose concentration in diabetic pets. They compared glucose measured in blood obtained from the inner pinna using a lancet device, with serum glucose measured in jugular blood.¹⁰ The authors’ study, however, is the first one to propose an alternative blood sampling method in dogs for common plasma and hematological examinations.

One question that arises from the authors’ results is: Does the M technique induce alteration in plasma and hematological variables in comparison to the conventional V technique? Ten of the 24 variables tested showed a statistically significant effect, and all (glucose, ALT, WBC, eLC, eGC, RBC, Hb, Ht, and NC) were overestimated by M except EC and PCV. The reasons for these differences were not established.

Hypotheses evoked in previous studies to explain differences between marginal ear vein and cephalic or saphenous blood in the feline species (such as a plasma skimming effect in small vessels or accumulation of a higher density of blood cells toward the central axis of the vessel with increasing blood flow rate^11,12) were not relevant here.

The differences observed between the two techniques in the present study may be attributable to sampling site as well as sampling methodology. Whatever their origin, their amplitude appears too slight to merit further attention. Ideally, the difference between the values obtained with M and those obtained with V should be interpreted by comparison with the critical difference. The critical difference for a given variable is calculated from the between-subjects variance, the within-subject variance, and the analytical variance. This parameter may help to ascertain whether the difference between two consecutive measurements can safely be ascribed to natural (i.e., clinical) variations or not. If the difference between two consecutive measurements is less than the critical difference, then it may be ascribed to random variations.

Of the variables for which a significant effect of sampling technique was observed here, a critical difference has been described only for glucose and ALT (1.49 mmol/L and 13.3 U/L, respectively).¹³ Although defined with other analytical methods and in dogs showing other characteristics, these values may be used to interpret the potential clinical relevance of differences due to sampling technique. For plasma glucose, all the differences between M and V were <1.49 mmol/L. For ALT, only four out of 14 differences between M and V were >13.3 U/L. The maximal difference observed was 26 U/L in one dog, but the median value was 2.5 U/L [Table 2]. The authors can therefore consider that the use of M instead of V would not likely lead to clinical misinterpretation of the results.

No critical difference values have been established, to the authors’ knowledge, for hematological variables in the dog. However, the relevance of the difference in test results can be graded to some extent by comparing the median value of the difference to the corresponding canine reference interval.⁹ Comparison of the maximal difference observed and the median of the differences between the two techniques for each variable with the reference interval showed that the differences would not likely lead to misdiagnosis [Table 2]. Moreover, the median values of the differences and the maximal differences observed for Hb, WBC, eLC, and NC counts in this study were lower than those observed in a previous study. In that previous study, blood samples taken from the cephalic and marginal ear veins in cats were compared, and the observed differences in test results were concluded to be of only minor practical importance (data not shown).¹²

Results obtained from blood films should be interpreted with caution because of the inherent imprecision in blood film preparation. The coefficients of variation for LC and MC were relatively large (from 40% to 68%), and this could explain why no difference due to sampling technique could be seen.

Several potential drawbacks in the experimental design of the present study must be mentioned. First, only seven dogs were used, which could appear as too few, especially in view of the number of animals (¡Ý40) currently recommended for the validation of analytical methods.^14–16 Nevertheless, validating an analytical method was not the aim of the present study. Although not ideal, the sample size in this study proved to be large enough, because it was possible to evidence small, significant differences between M and V for some variables. A distinct advantage of this study was that all dogs underwent both sampling techniques twice on the same day with an inverse sequence (M-V and V-M) in the morning and in the afternoon, which allowed 14 comparisons to be made. Also, no period effects (i.e., morning versus afternoon results) were observed except for ALP and the eEC differential count estimated by the analyzer. Moreover, the conditions were very well controlled. The same well-trained investigator conducted all samplings and used both methods; the same assistant was used for restraining the dogs; and the same laboratory analytical conditions were used for all the assays with a well-trained technician).

Another limitation of the present study is that only healthy, medium-sized dogs were used, whereas such a sampling alternative would probably be more helpful in miniature-breed dogs. Alternative blood sampling methods are indeed designed to improve practicability, tolerance, and safety of this routine veterinary procedure. In this study, sampling blood with the V technique was found to be as well tolerated and safe as the M technique. However, the dogs used were cooperative experimental beagles, and in the authors’ strong opinions based on experience, jugular venipuncture can prove to be more difficult and hazardous than blood sampling from the cephalic vein in everyday practice. Moreover, because the animals were healthy, it was impossible to identify issues that might arise with capillary filling. For example, no dogs suffered from specific pathological conditions (such as dehydration, hypotension, or shock) that compromise peripheral circulation. Experimental healthy beagles were selected here, as it would have been difficult to get the owner’s consent to perform two successive venous punctures in the morning and in the afternoon on the same animal.

The ranges of values for tested variables were also limited, except for creatinine (discussed later). A similar comparison of both methods in a group of diseased animals with a wider range of values would have been appropriate. However, using healthy dogs seemed to be the only way to ensure the well-controlled conditions achieved in this study (i.e., same investigators, same assay [method and batches], all samples processed within the same day). Moreover, when measures are repeated in clinical conditions, fluctuations of the tested variables may occur spontaneously and independently of the sampling technique due to the course of the disease. The authors’ results show that for plasma creatinine, both sampling techniques gave similar results over a range of values that covered those observed in clinical settings. Very high plasma levels were obtained after the administration of exogenous creatinine, and no difference was observed between sampling techniques.

The similar results for the plasma exogenous creatinine clearance test show that blood sampled from a cephalic vein with a capillary system may readily be used for serial testing and the assessment of exponentially decreasing concentrations of an analyte (as performed in pharmacokinetic studies). When repeated blood sampling is required, a catheter may also be used, but this requires a strict aseptic technique and discarding the blood volume that corresponds to the dead space at each sample time.¹⁷

Conclusion

These results are encouraging, as they show that a pediatric device using a capillary system provides similar results to those obtained with the vacuum tube technique in healthy, medium-sized dogs. Nevertheless, a difference between techniques for values outside the range observed here cannot be excluded. Although unlikely, technical sampling issues in smaller dogs or those associated with peripheral circulatory compromise cannot be fully ruled out either. Further investigations should therefore be conducted in larger populations of dogs before the use of M can be definitively recommended.