Cephalic and saphenous venous blood collected by continuous heating of the paws compared with arterial blood for measurement of blood gas values in well‐perfused dogs

Abstract Background “Arterialization” of the dorsal hand vein is well‐established in human medicine, but not in veterinary medicine. Objectives To compare cephalic and saphenous venous blood collected by continuously heating the paws to 37°C (“arterialization”), with arterial blood (AB) for measurement of blood gas variables in well‐perfused dogs. Animals Eight healthy dogs. Methods Experimental study. Fore and hind paws were continuously heated to 37°C to “arterialize” cephalic and saphenous venous blood. AB and “arterialized” cephalic and saphenous venous blood (ACV and ASV, respectively) were simultaneously collected from lightly anesthetized dogs with induced metabolic and respiratory acid‐base disorders. The pH, partial pressures of carbon dioxide (PCO2) and oxygen (PO2), bicarbonate concentration [HCO3 −], and base excess (BE) were measured once in each state. Systolic blood pressure was maintained above 100 mm Hg. The AB, ACV, and ASV values were compared. Results The pH, [HCO3 −], and BE values had no significant difference and good agreement, the PCO2 values had a strong correlation (correlation coefficient of .91‐1.00), and the PO2 values had a significant difference (P < .01) and poor agreement between AB and ACV, and between AB and ASV. The PCO2 values of ASV overestimated those of AB by ~3.0 mm Hg, which was considered within clinically allowable limits, while those of ACV were not within clinically allowable limits. Conclusions and Clinical Importance Under experimental conditions, the ASV samples were more identical to the AB samples than the ACV samples for pH, PCO2, [HCO3 −], and BE values in well‐perfused dogs. The saphenous vein is suitable for “arterialization.”


| INTRODUCTION
Venous blood samples have been used to estimate acid-base balance in human [1][2][3][4][5][6][7][8][9] and veterinary medicine, [10][11][12][13] instead of arterial blood (AB) samples. However, because of its inaccuracy, venous blood gas analysis might be inadequate for clinical decision-making. 14,15 In human medicine, "arterialization" of the dorsal hand vein is a well-established blood sampling technique. In well-perfused humans, heating hands to 42 C to 43 C for 10 to 15 minutes [16][17][18] causes venous blood to become more similar to AB. 1,19 This technique allows sample collection without venous stasis and with the patient lying in bed. 1,20 Therefore, "arterialization" is an easy, safe, and noninvasive technique for AB gas sampling. In humans, "arterialized" venous blood can substitute AB for the measurement of pH, partial pressure of carbon dioxide (PCO 2 ), and lactate. 1,16,20,21 In modern human medicine, "arterialization" has only been applied to volunteer subjects participating in medical research. 17,22,23 Because this time-consuming procedure before sampling prevents collecting samples when they are urgently needed, it is rarely used in clinical practice. Furthermore, heating the hand to temperatures above 42 C might be uncomfortable for some patients. 1,21 However, adequate "arterialization" can be achieved as long as the cutaneous temperature remains above 35 C to 37 C. 1,17 To our knowledge, only 1 experimental study has documented the use of "arterialized" venous blood in dogs, 24 in which the cephalic vein was "arterialized" by continuously heating the dogs' forepaws to 40 C using an incandescent lamp. It might be possible to collect blood as necessary without causing discomfort by continuously heating the forepaw to a relatively low cutaneous temperature of 35 C to 37 C.
"Arterialized" peripheral venous blood gas analysis might be useful for repeated blood gas sampling in animals with severe uremic acidosis and diabetic ketoacidosis, 25 as well as in animals under postoperative observation. In veterinary clinical settings, the cephalic vein is often used for intravenous infusions and is not always an appropriate site for "arterialized" venous blood sampling. The saphenous vein in the hind leg, which is used for venous blood sampling as an alternative to the cephalic vein, is a preferred site for "arterialized" venous blood sampling. However, information on "arterialization," including suitable sites for venous blood sampling, is lacking in veterinary medicine.
The present study aimed to compare cephalic and saphenous venous blood collected by continuously heating the paws to 37 C ("arterialization") with AB for the blood gas and acid-base values in wellperfused dogs with metabolic and respiratory acid-base disorders.

| Animals
This study was approved by the institutional laboratory animal care and use committee of the study institute. In this study, eight clinically healthy adult crossbreed dogs (4 males and 4 females; KITAYAMA LABES CO., LTD., Nagano, Japan) were used. Their median age was 2.0 (range, 2.0-6.0) years; their median body weight was 8.5 (range, 7.6-13.1) kg; their median packed cell volume was 39.4% (range, 34.5%-52.7%); and their median hemoglobin concentration was 13.9 (range, 12.2-19.1) g/dL. Physical examination, chest X-rays, a complete blood count, and a blood biochemistry profile revealed that all of the dogs used in the study showed no indication of cardiovascular and respiratory ailments.

| Study design
The dogs were lightly anesthetized and maintained in a normal metabolic and respiratory state while breathing air until the first experiment, in which metabolic acidosis and alkalosis were induced.
Following appropriate intervals to allow the washout of induced acidbase disorders, the dogs were allowed to return to their normal acidbase state before the second experiment, in which respiratory acidosis and alkalosis were experimentally induced. Similarly to the first experiment, appropriate intervals were maintained in the second experiment to allow washout of the acid-base disturbed states, and the dogs were allowed to return to their normal state. The order of the induced acidbase states was randomized (randomized block design). 26 The present study consisted of eight blocks that were patterns of orders of induced acid-base states, with one dog allocated to each block; thus, a minimum of eight dogs were required in the present study ( Figure 1).
Before the experiments, two preliminary experiments were performed in two normal, healthy adult dogs to standardize the study protocol and washout intervals based on a previous study's protocol. 24 Steady heat was applied to the fore and hind paws of the dogs using two incandescent lamps set 10 to 15 cm lateral to the paws to maintain a cutaneous temperature of 37 C to intend to "arterialize" cephalic and saphenous venous blood. AB, "arterialized" cephalic venous blood (ACV), and "arterialized" saphenous venous blood (ASV) samples were collected simultaneously once in each acid-base state and analyzed for pH, PCO 2 , partial pressure of oxygen (PO 2 ), bicarbonate concentration ([HCO 3 À ]), and base excess (BE). The values of the ACV and ASV samples were compared with those of the AB samples. The overall ambient temperature of the experimental setup was maintained between 22 C and 24 C. During blood sampling, three co-experimenters, all of whom were professional veterinary practitioners, were randomly allocated to each sampling site. They were not informed about the interim report on experimental results during the experiments.

| Anesthesia and maintenance
All dogs were pretreated with acetylpromazine (0.1 mg/kg IM), butorphanol (0.2 mg/kg IM), and atropine (0.05 mg/kg IM), followed by anesthesia with thiamylal (10 mg/kg IV). The dogs were intubated and allowed to breathe ambient air while lying in lateral recumbency on an electric heating blanket. Based on previous studies, 27-30 a continuous infusion of ketamine (10-60 μg/kg/min), pentobarbital (5 mg/kg IV, appropriately q1-3h), and butorphanol (.05 mg/kg IM, q1h) were administered to ensure the dogs were on a light plane of anesthesia throughout the experiments without affecting cardiorespiratory function or losing eyelid reflex. The infusion was maintained via a cephalic vein that was not used to collect blood samples during the experiments. Two thermometer probes were surgically inserted into each dog's fore and hind paws to record cutaneous temperature.
The oxygen saturation (SpO 2 ) was measured with a pulse oximeter (CAPNOX, Colin, Aichi, Japan), the breathing rate and end-tidal PCO 2 were measured with a side-stream capnometer (CAPNOX, Colin, Aichi, Japan), and the AB pressure was measured with an oscillometric blood pressure device (Dynamap 8300, Critikon, Tampa, FL). The heart rate and rhythm, as well as the rectal and esophageal temperatures, were continuously monitored. When the dogs returned to a steady state, the baseline values of all variables and the tidal volume were recorded using a respirometer (RM121, Citizen, Yamanashi, Japan). SpO 2 was monitored and maintained at or above 90%. To avoid severe hypoxemia, the dogs were given 100% oxygen if their SpO 2 fell under 90%. Systolic arterial pressure and heart rate were maintained above 100 mm Hg and between 70 and 150 beats/min, respectively, by adjusting the rate of ketamine infusion, as appropriate. Rectal and esophageal temperatures were maintained around 38 C by adjusting the temperature of the electric heating blanket.

| Blood sample collection, handling, and measurement
AB samples were collected from the femoral artery in heparinized syringes through a 22-gauge catheter with an extension tube and a 3-way stopcock. ACV and ASV samples were collected from the distal part of the limb via venipuncture with minimal venous stasis to avoid blood flow obstruction. All three blood samples (2 mL each) were obtained anaerobically. Before blood sampling, vital signs, SpO 2 , and end-tidal PCO 2 were maintained stable and steady for at least 5 minutes. The syringe tubes were immediately and securely capped, placed on ice, and analyzed within 10 minutes in a random sequence on a blood gas analyzer 31

| Experiment 1: Metabolic acid-base disorders
All arterial variables were confirmed to be within the normal range before any experiments. Based on these initial baseline values, mechanical ventilation (Compos β-EV, Silver Medical, Tokyo, Japan) with compressed air (Kawasaki Sogo Gas Center, Kawasaki, Japan) was initiated at a frequency of 8 to 16 breaths/min and a tidal volume of 10 to 25 mL/kg. This state, that is, Normal 1, was maintained for 10 minutes before simultaneous blood sampling. After sampling, the dogs were allowed to breathe ambient air without mechanical ventilation.
Metabolic acidosis was induced by infusing 0.6-M ammonium chloride (NH 4 Cl; Conclyte-A, Hishiyama Pharmaceutical, Osaka, Japan) in a 5% dextrose solution at a rate of 3.5 to 6.5 mEq/kg/h for 60 to 100 minutes before simultaneous blood sampling. To induce metabolic alkalosis, 1 M sodium bicarbonate (NaHCO 3 ; MEYLON84, Otsuka Pharmaceutical Factory Inc., Tokushima, Japan) was infused at a rate of 5 to 10 mEq/kg/h for 60 to 100 minutes before simultaneous blood sampling. During Experiment 1, the acid-base state was monitored, and AB gases were measured at 15-minute intervals. To allow for washout, a 2-hour interval was allowed between the metabolic acidosis and metabolic alkalosis states. All dogs were allowed to recover from metabolic acidosis and metabolic alkalosis with appropriate treatments.

| Experiment 2: Respiratory acid-base disorders
Normoxic ventilation was established, and blood samples from the three sites were collected simultaneously in this state, that is, Normal F I G U R E 1 Repeated measures and a randomized block design in the present study. All types of acid-base disorders were experimentally induced. Ten minutes before producing metabolic or respiratory acid-base disorders, the acid-base state was controlled normally. Blood samples were collected once in each state. It took 1 to 3 hours to wash out between these acid-base states. These six states could be randomly arranged in eight blocks. One dog was allocated to each block. Therefore, eight experiments were completely performed.
2, as described in Experiment 1. Next, respiratory acidosis was induced by mechanical ventilation with 100% oxygen at a frequency of 2 to 4 breaths/min and a tidal volume of 15 to 25 mL/kg. To impede hypoxemia, 100% oxygen was used. To induce apnea and adjust the respiratory rate, succinylcholine (2 mg/head IV; Succin, Yamanouchi Pharmaceutical, Tokyo, Japan) and pentobarbital (2.5 mg/kg IV) were administered at 15-minute intervals. Blood samples were collected after a 60-minute treatment period. To induce respiratory alkalosis, mechanical ventilation with compressed air at a frequency of 10 to 16 breaths/min and a tidal volume of 25 to 50 mL/kg was used. Mechanical ventilation was maintained for 60 minutes before blood sampling. To maintain good perfusion, half saline (saline:5% dextrose, 1:1) was infused at a rate of 5 to 10 mL/kg/h throughout Experiment 2.
When Experiment 1 was followed by Experiment 2, the dogs were given a 3-hour washout interval. When Experiment 2 was followed by Experiment 1, the dogs were given a 1-hour washout interval. Following all experimental procedures, the dogs were allowed to extubate and awaken from anesthesia.

| Statistical analysis
To determine the validity of the washout, mean differences in the values of all arterial variables between the Normal 1 and 2 states were calculated, and the arterial values between these two states were compared using a two-tailed paired Student's t-test.
The mean values of all variables in arterial and "arterialized" venous blood samples during all the induced acid-base states were compared using a multivariate approach to repeatedmeasures analysis of variance (MANOVA). 32 The strength of the linear relationship between arterial and "arterialized" venous blood sample variables during induced metabolic and respiratory acid-base states was examined using linear regression analysis, and correlation coefficients (R) and coefficients of determination (R 2 ) were calculated. The degree of agreement between arterial and "arterialized" venous blood sample variables was assessed by plotting the differences between the two measurements against their average (ie, Bland-Altman plots 33 ). The PO 2 data were divided into two groups of states during oxygen inhalation at fraction of inspiratory oxygen (FIO 2 ) values of 1.0 and 0.21, which have a significant impact on PO 2 . All data were analyzed using the Statistical Package for the Social Sciences (IBM SPSS Statistics, Version 28.0, Armonk, NY). A P-value of <.05 was considered statistically significant.
In the present study, clinical agreement was determined by defining the clinically allowable limits for each variable on Bland-Altman plots using the following criteria required to pass proficiency testing for quality control 2,34-36 : 80% of the data points should be within ±0.04 for pH, ±5.00 mm Hg for PCO 2 , and ±15.51 mm Hg for PO 2 on a standard analyzer. 35,36 The clinically allowable limits for [HCO 3 À ] were set at ±1.50 mmol/L to account for the analyzer's accuracy. 31 3 | RESULTS

| Heated-paw technique
At the beginning of the experiments, the median cutaneous temperature of the fore and hind paws was 35.2 C (range, 32.1 C-37.0 C) before applying the heat lamp, and the paws were heated to a cutaneous temperature of 37 C using incandescent lamps to intend to "arter-
No significant differences were detected between the two states, except for BE. The mean differences in BE were clinically insignificant.
Therefore, the washout intervals were considered valid.

| Vital signs and AB pressure
During this experiment, the heart and respiratory rates, rectal and esoph-    Figure 5A

| PO 2
PO 2 values in both the ACV and ASV blood samples differed significantly from those in the AB samples (P < .01; Table 1). The measurements did not correlate, and the data points were not within the clinically allowable limits (4.2%-8.3%) with AB during O 2 inhalation at FIO 2 of 0.21 and 1.0 under all acid-base states (Figures 6 and 7).  15,48,50,51 Arterial catheterization is also at risk of hemorrhage, 15 thrombosis, 15 infection, 15 and, although rarely, severe vascular occlusion 15 or fatal arterial rupture. 52 Both arterial puncture and catheterization are difficult to perform in small or obese animals. 31,52 "Arterialization" is a fascinating theory for obtaining venous samples The AB gas and acid-base values in the present study are similar to those reported in previous studies in healthy, awake dogs, 11,12 indicating that the experimental paradigm used in the present study provided an environment that closely resembled that of dogs in the awake state. In general, the average PCO 2 value is around 40 mm Hg in normal humans and slightly lower in dogs (37 mm Hg). 47 The present study results also supported these findings.  "Arterialization" using the heated-paw technique in the present study is also considered to minimize the local tissue metabolic effect.

| DISCUSSION
Our findings are consistent with those of human studies. 1,16,22,53,54 Our findings are also consistent with those of a study on dogs, which found a high correlation of pH, PCO 2 , and [HCO 3 À ] values between the AB and ACV samples. 24 In the present study, PCO 2 values of both the ACV and ASV samples were significantly different from those of the AB samples under  The present study revealed a strong, positive correlation of PCO 2 between the AB and ASV samples compared with that between the AB and ACV samples. Since the blood sampling site of the ASV is anatomically more distal to the limb than that of the ACV, the ASV is less affected by tissue metabolism than the ACV. In humans, the veins on the back of the hand are commonly used to collect "arterialized" blood. These veins drain tissues with low metabolism (primarily skin and bone) and are easily dilated by warmth to increase blood flow through the hand. 20 In dogs, the saphenous veins also meet this criterion.
Hypovolemia or anemia increases arteriovenous differences in acid-base states and affects blood gas values. 13 reliably for dogs in hypovolemic shock because of peripheral hypoperfusion. In these animals, AB gas should be analyzed to accurately assess oxygenation and acid-base balance.
Capillary blood extracted from toenails 44 and pinna margins 13 can also be used to measure blood gases as an alternative to AB sampling.
These sites are easier to access and have fewer complications. 12,13,44,45,58 However, these sites are rarely used as air contamination might occur during blood collection. 16 In comparison to capillary blood, peripheral venous blood sampling has the advantage of having no limit on the amount of blood collected, allowing for repeated analyses of many variables other than blood gas analysis, and avoiding air contamination. 16 Lingual venous blood, which can also be used as a good substitute for AB for measuring blood gases, 42 is not feasible in unanesthetized dogs.
In the present study, incandescent lamps were used to continuously heat paws, as in a previous study. 24 However, this method appears impractical because the paws cannot be heated if the animal stands up or moves. Based on the present study results, further studies should be conducted to develop a practical, stable, and safe device to maintain the cutaneous temperature at 37 C in the dorsal part of the paw. In human studies, electric warming pads with a maximum temperature of 60 C were used. 1,16,20 In these studies, the hand was heated by loosely bound pads that were separated from the skin by the sleeve of a bed jacket, and the cutaneous temperature was maintained at around 40 C for 10 to 15 minutes. 1,16,20 We believe this technique is applicable to dogs.
Ideally, a venipuncture needle 59 or cannula 60 is introduced retrogradely, with the point directed toward the fingers; however, many earlier investigators did not mention this. 1,16,17,20,21,24 In the present study, venipuncture was not performed retrogradely, but sufficiently "arterialized" venous samples were obtained. More research is required to explore the significance of needle direction.
The findings of our study should be interpreted in light of several other limitations. First, the present study did not include data on venous blood samples collected from the nonheated paw. Therefore, the effect of heating on "arterialization" was not precisely evaluated.
Additionally, general anesthesia might have strengthened the correlation and agreement between arterial and "arterialized" venous blood variables by reducing vasoconstriction via suppression of central thermoregulation, 20 thereby inducing spontaneous "arterialization" of venous blood. 1 Thus, in the present study, the strong correlation between the AB and "arterialized" venous samples might be because of the effects of multiple factors. However, the technique of continuously heating the paws appears to be the main factor, because a previous experiment in dogs under general anesthesia 24 62 ). Ketamine also causes mild but sustained vasodilation that is not dose-dependent. 63 In the present study, ketamine was administered at a rate of 10 to 60 μg/kg/min. As expected during our experiments, heart rate, systolic AB pressure, and mean arterial pressure were all close to or within the reference ranges in normal dogs (70-120 beats/min, 120 mm Hg, and 87 mm Hg, respectively). 64 The present study provided an ideal environment for collecting "arterialized" venous blood. Nevertheless, the PCO 2 of the ASV had a systematic bias of +3 mm Hg when compared with the arterial value.
The difference was within allowable limits. However, if bias is considered critical, the acid-base status should be determined using AB samples or data calibrated to account for a bias of +3 mm Hg.
In conclusion, under experimental conditions, the ASV samples were more identical to the AB samples than the ACV samples for pH, ing. This method might be useful in cases where ketamine infusion is used for postoperative analgesia.

ACKNOWLEDGMENT
No funding was received for this study. This study was presented in part as an oral presentation at the 2012 American College of Veterinary Internal Medicine Forum, New Orleans, LA. We are grateful to Sysmex in Kobe, Japan for providing the blood gas analyzer and accompanying kits necessary to conduct this study.