SUBJECTS

Twenty premature infants were recruited from the Section of Neonatology, Department of Paediatrics, the National Hospital, Oslo. Infants who were healthy, without fever, not suffering from intraventricular haemorrhage, had not received analgesics or sedatives within the past 48 hours, and not exposed to nasal continuous positive airway pressure intervention were eligible for participation in the study. There were 13 girls and seven boys. The infants were born between 29 and 35 weeks (median 33 weeks) gestational age and at the time of the study were between 1 and 25 days postnatal age (median 7 days). None had reached 37 weeks corrected gestational age. Median weight at birth was 2000 g (range 1282–2550), and at the time of participation in the study the median weight was 1930 g (range 1190–2390). Five minute Apgar scores had a median of 8.5 (range 7–10). To investigate how the number of previous heel sticks influenced the skin conductance activity variables, the number of times doctors had previously ordered blood samples for the infants were examined. It was between 0 and 35 times (median 11). If only a blood glucose test had been ordered, this was not included because the tissue injury made to obtain blood for this test is only minor. Skin conductance activity was measured when the infants were exposed to heel stick for routine blood sampling. Informed parental consent was obtained before inclusion of an infant in the study. The international ethics committee approved the study.

APPARATUS

Skin conductance activity was measured by alternating current at 88 Hz.10 Low frequency electrical conductance reflects the ionic conduction in the stratum corneum, which is largely determined by sweat duct filling.11 ,12 A frequency of 88 Hz is sufficiently high to reduce the requirements for low electrode polarisability considerably, but also low enough to ensure minimal influence from layers other than the stratum corneum.12 An applied voltage of 50 mV and a three electrode system were used.13 The three electrode system comprises a measuring electrode, a counter current electrode, and a reference voltage electrode, which ensures a constant applied voltage across the stratum corneum beneath the measuring electrode.14

The apparatus (fig 1) conforms to the safety regulations given in IEC 60601. Beckman electrodes (Sensormedicis, Iorba Linda, California, USA) were used. The electrodes were attached to the skin by disks of double sided adhesive tape from 3M, Minneapolis, Minnesota, USA. Conductive paste from the National Hospital Pharmacy, Oslo, Norway, containing 6 g hydroxyethylcellulose 700, 0.58 g NaCl, 0.1 g methylparahydroxybenzene, 0.1 g propylparahydroxybenzene, 2 g alcohol 96%, distilled water up to 100 g, was used to improve electrode conductance.

• Download figure
• Open in new tab
• Download powerpoint

Figure 1

Diagram of apparatus used to measure skin conductance activity. Reproduced with permission from Asbjørn Fremming (postgraduate thesis, Institute of Physics, University of Oslo, 1998).

SOFTWARE PROGRAM

The data were stored on line using a portable computer (Compaq Armada) and were analysed off line with a software analysis package. The sample frequency was 50 Hz and the resolution was 12 bits. The software analysis program for skin conductance activity was carried out in Labview, National Instruments, USA, and was developed by us. The program recorded and counted the number of waves per second, by defining the valleys and peaks, and calculated the mean of the amplitudes of the waves and the mean baseline in the study period for the spontaneous skin conductance activity. The valleys and peaks were established when the derivative of the wave was 0. The amplitude of the wave was calculated from the bottom of the valley before the peak to the height of the peak. The slope was defined as (the mean distance of the valley to the peak)/(time to reach peak) (fig2).

• Download figure
• Open in new tab
• Download powerpoint

Figure 2

(A) Spontaneous skin conductance activity (SCA) measured before, during, and after painful stimulation in a premature infant born at 29.1 weeks gestational age who had lived for 2 days. (B) The stress response in (A), from 166 to 184 seconds, expanded to show the valleys and peaks in detail.

The program contained a function that enabled us to define a threshold for these values. To eliminate electronic noise, the definition of minimum amplitude was set at 0.02 μS. Moreover, to eliminate artefacts, the slope was set at less than 2 μS/second. The width of the waves was unlimited. Artefacts occurred if an electrode became detached from the skin. The method was not sensitive to movements or changes within normal room temperature. The software analysis program could also analyse smaller segments of the registered data. This function was used if artefacts were found. Moreover, in order to examine details of the registered data, a particular time period during registration could be chosen and expanded.

The apparatus and software program have been commercially developed by Med-Storm Innovation, Oslo, Norway, product number 060895.

PROCEDURE

Testing was conducted in the intensive care area or the intermediate area at the Section of Neonatology. The infants were subjected to heel lancing for routine blood collection to test for metabolic disease and physiological function. The standard protocol for sampling blood involved warming the left foot, picking it up, making a small incision with a metal scalpel (2.2 mm long and 1 mm wide), squeezing the heel, and if necessary repeating the incision to draw sufficient blood. Blood was collected for at least the following tests: haemoglobin, leucocytes, platelets, C reactive protein, Na⁺, K⁺, Ca²⁺, acid-base.

Electrodes were fastened to the infant's right foot 10 minutes before the heel stick was scheduled. The counter current electrode was placed on the medial right side of the foot over the abductor hallucis muscle adjacent to the plantar surface, the measuring electrode was placed midway between the first phalanx and a point directly beneath the ankle, and the reference voltage electrode was placed on the dorsal side of the foot. These areas fit with the Edelberg guidelines for the placement of electrodes in order to obtain the most sensitive measurement.16 All the heel pricks were scheduled between 0800 and 1000, at least one hour after feeding. A third of the infants received 2 ml 15% oral sucrose before heel stick. Behavioural state and skin conductance activity were measured for three minutes before heel stick, during the heel stick and squeezing period, and for three minutes after the squeezing period was finished (behavioural state: 0, non-REM sleep; 1, REM sleep; 2, awake and calm movements; 3, awake and very active movements; 4, crying). The heel stick and squeezing period lasted for 90–140 seconds.

STATISTICAL ANALYSIS

To test the stress response during heel stick, the skin conductance activity variables and behavioural state were analysed by paired non-parametric tests before, during, and after the procedure. First the Friedman test18 was used to look for significant values in the three variables (before, during, and after heel lance). If statistically significant values were found with the Friedman test, the Wilcoxon test was used to compare the levels before the heel lance with those during and after the heel lance. The Spearman correlation test was used to study whether behavioural state during heel prick correlated with the skin conductance activity variables.

In addition, a linear regression model was used to study how gestational age, postnatal age, and the number of times blood samples had been ordered influenced the skin conductance activity variables and the behavioural state during heel lance. To determine which factor had the greatest influence on the skin conductance activity variables and the behavioural state during heel lance, a multiple stepwise regression analysis was performed on the statistically significant variables.19 All the statistical tests were performed using SPSS 8.0.