2.1

Experimental procedure

The present study was conducted in accordance with The Helsinki Declaration and approved by Ethics Committee of the University of Latvia. Prior to the study, every subject was informed about all procedures, particularly emphasizing on the possible discomfort during long occlusion and of blood sampling procedure from the fingers. Thirteen healthy subjects (25 ± 5 y.o., mean±SD) gave informed consent and were enrolled in the study.

To insure adequate microcirculation, the surface temperature of the right middle and index fingers was measured with a contactless thermometer (Raytech Inc.) and subjects with the skin temperature below 27°C, were excluded from the study because of reduced cutaneous blood flow and possibly compromised baseline skin saturation.

Before experimental procedure, subject was seated in a comfortable chair with supported hands. The entire protocol comprises four consecutive stages, see Fig.1.A. During the first stage, an intact finger (second index finger) was disinfected with isopropyl alcohol pad and pricked using finger picker (soft click, Accucheck). The small capillary blood sample (120uL) was collected using heparinized sampling tube (Accriva Diagnostics) and transferred to CG4+ cartridge for analysis with a portable whole blood analyzer (iSTAT, Abbott).

Figure 1.

Finger arterial occlusion protocol (A) and acquisition of skin hyperspectral cube (B). Blue color indicates occluded finger (3), red color-intact finger (2).

In order to produce alterations of skin capillary blood biochemical parameters and oxygen saturation, in the second stage of protocol the proximal phalanx of the right middle finger was occluded with a pneumatic cuff at the 150 mmHg pressure and kept occluded for 25 minutes.

Seemingly long finger arterial occlusion time is common in clinics and laboratory settings and not harmful to subjects. Performed as a routine during orthopedic operations; with the occlusion time around 2h and the cuff pressure 50 mmHg above patient systolic blood pressure. The recommended single occlusion time should not exceed 30 minutes for elderly and critically ill patients⁵, following the 3-5 min post-occlusion period prior to next occlusion⁶.

There are several papers describing normal physiological studies (healthy volunteers) with the limb occlusion up to 30 minutes at suprasystolic cuff pressure without any harm to subjects ^7–10. Moreover, in our study, subjects were able to interrupt occlusion procedure at any moment.

In the third stage of protocol during the last minute (25^th) of occlusion, the hyperspectral cube (HIS cube) from the hairy skin of both occluded (middle) and intact (index) finger was captured at 450nm-800nm range and 5nm spectral resolution with tunable filter hyperspectral camera, Nuance EX (CRi, UK) as depicted in Fig.1.B. The HIS cube acquisition procedure has been described in detail elsewhere¹¹.

Within the minute after hyperspectral acquisition, capillary blood was collected from the occluded finger and analyzed by the same portable analyzer-fourth stage. After that, pneumatic cuff has been released and the finger returned to the normal state within the following 1-3 minutes.

The six tissue ischemia characterizing blood parameters were obtained from whole blood analyzer from intact and occluded finger: pCO₂-carbon dioxide partial pressure, pO₂-oxygen partial pressure, Lact-lactate concentration, pH-acidity, HCO³- bicarbonate concentration and SaO₂- Hemoglobin saturation with the oxygen.

2.2

The processing of hyperspectral data

Oxygen saturation was calculated using light diffusion model in the skin^12,13.

In the modeling process it has been assumed that skin consists of three layers: stratum corneum (SC), epidermis (epid.), and dermis (derm.). Another assumption was that SC does not absorb light, only providing scattering of the light as it contains mainly keratin. Epidermal layer has skin pigment melanin which absorbs light and the main absorber in the dermal layer is blood, which can be divided in absorption of oxy/deoxy-hemoglobin (HbO₂ and HbD).

Accordingly, absorption coefficients are:

where C_mel, C_hbO2, C_hbD is chromophore concentration in skin, ε_mel, ε_hbO2, ε_hbD is extinction coefficients of melanin, HbO₂ and HbD.

The scattering occurs in whole skin, each layer has different scattering properties. The model is sophisticated as the scattering depends also on absorption in skin chromophores¹⁴:

where µ_s500 is reference scattering at λ = 500nm, ρ, γ – scattering parameters. Attenuation coefficient is µ = µ_a+µ_s.

Using 3-layer light diffusion model, we obtain reflectance spectrum R(λ) of the model.

Reflectance depends on C_mel, C_hbO2, C_hbD and model parameters µ_s500sc, µ_s500epid, ρ_sc, ρ_epid, γ_sc, γ_epid and layers thicknesses. Chromophore concentrations are found fitting reflectance spectrum of the model to measured reflectance. Since model parameters can slightly deviate from expected [3], we allow change in these parameters within a small range using fitting.

The final skin microcirculation parameter is oxygen saturation (SaO₂), which can be found from the following equation:

For off-line analysis of HSI data acquired by Nuance camera, image processing software was developed in the Matlab GUI environment (Fig. 2). This software allows visualization of oxygen saturation maps, mapping its distribution in different skin regions of different body parts. Computation of maps is performed by algorithmic steps described in our previous study ¹¹.

Figure 2.

The screenshot of software interface for hyperspectral data analyses and blood saturation mapping. The fingers photo was digitally contrasted to emphasize skin color tone diferences.

2.3

Statistical analyses

The statistical analysis was performed using (SigmaPlot12, Systat Software) statistical software. The group values were expressed as the arithmetic mean ±standard deviation. To compare the group mean values, Student-t test was utilized, considering p<0.005 significance level. The comparison of methods (HIS vs REF blood analyzer) was performed utilizing Bland-Altman Analyses.

3.1

Capillary blood biochemical parameters

The baseline values of capillary blood parameters in the intact finger were very similar for all subjects, displaying small standard deviation values, arithmetic mean ±standard deviation values are as follow: pCO₂- 36.77±3.95 mmHg, pO₂-74.20±5.97 mmHg, Lact-1.72±0.85 mmol/L, pH- 7.44±0.03, HCO³- 24.93±2.07mmol/L and SaO₂- 95.13±1.46 %.

The obtained values were slightly lower than typical arterial values indicating normal metabolism, cardiopulmonary function and optimal cutaneous blood supply ¹⁵¹⁶. The obtained capillary blood hemoglobin saturation values were in the physiological range, hence slightly lowered in comparison to arterial blood.

Taking into the consideration sufficient cutaneous perfusion, as confirmed by skin temperature measurement, and relatively small volume of fingers, the values of these blood parameters can be attributed to the cutaneous circulation, which is partly determined by superficial vascular plexus situated in papillary dermis.

Similar baseline values for oxygen and carbon dioxide concentrations have been reported during transcutaneous gas measurement: tcO₂=73.9±14.6 mmHg and tcCO₂=40.5±9.8mmHg supporting our findings¹⁷. Our assumption is advocated by the observation of local differences between capillary blood values from different sites of the body¹⁸. The different tissue has different energetic metabolism, perfusion and capillary density¹⁹. The hands and fingers are unique as they have very high capillary density and majority of capillaries are situated in the skin papillary dermis (approx. 7 times difference in density to other body sites), close to the skin surface¹⁹. The dermis exhibits an extensive vasculature that is arranged in two layers that are parallel to the skin surface. The superficial plexus is situated between the papillary and the upper reticular dermis, while deep plexus in the lower reticular dermis, and are connected by perpendicularly orientated communicating vessels, the capillary loop rise upwards into the papillae from the subpapillary plexus²⁰. The major determinant of cutaneous oxygen consumption is metabolism, which occurs in all layers of epidermis and dermis except stratum corneum. The oxygen demand is partially covered by the capillary blood supply and partly by diffusion from the atmosphere through the skin’s upper surface. In contrast, the epidermis has no vasculature, but is exposed directly to the atmosphere⁴.

Our approach on alteration of skin blood saturation is based on induction of hypoxia to the skin and underlying tissue by arresting blood supply, which should substantially change capillary blood parameters in occluded tissue. As expected earlier, twenty five minutes of finger arterial occlusion produced significant alterations of blood parameters proximally to occlusion site in all subjects, resembling blood gas profile seen in the clinics during severe acidosis and hypoxemia¹⁵²¹. The mechanism of occlusion is well known, as occlusion is utilized as a routine procedure during orthopedic surgery^22–24. During arrested arterial blood supply and venous blood drainage, occluded tissue try to maintain metabolism resulting in utilization of blood oxygen and production of carbon dioxide. After diminishing oxygen reserves, tissue energetic metabolism switches to anaerobic, substantially increasing acidity and concentration of anaerobic byproducts. Finger occlusion produced significant alterations of capillary blood for all 13 subjects indicating acidosis and hypoxemia in the occluded region. The observed blood values were as follow: pCO₂- 73.83±13.40 mmHg, pO₂- 21.93±4.62 mmHg, Lact-6.60±1.22 mmol/L, pH- 7.24±0.09, HCO³- 23.20±2.28mmol/L and SaO₂- 22.73±9.09 %, demonstrating larger dispersion of blood values in comparison to the baseline, possible because of different metabolic rate and size of the occluded tissue. Similar data has been obtained in occlusion in other studies, confirming our results²⁴. The comparison of group mean values in intact and occluded finger capillary blood parameters are depicted in Figure 3.

Figure 3.

Changes of capillary blood parameters during baseline (in intact finger) and occlusion (in occluded finger), reported values are group mean ± standard deviation, statistically significant difference (p<0.05) is denoted with asterisk

The observed capillary blood changes in occluded region – occluded finger last for less than a minute, following release of occlusion cuff, without any harm to the subjects. Because of relatively short occlusion time, small occluded tissue volume and relatively high capacity of peripheral tissue (finger) to adapt for hypoxia²⁵. The significant blood hemoglobin saturation drop has been achieved in the occluded finger as expected. Hence, occlusion influenced other undesirable blood parameters such as concentration of lactate, partial pressure of carbon dioxide and pH, which is considered as the side effect in our finger occlusion model, cannot be eliminated, due to the nature of physiological regulation mechanisms.

3.2

Skin hyperspectral parameters

During first minutes of occlusion, subjects felt slight discomfort which almost disappeared after 4-7 minutes following application of occlusion cuff and at the end of occlusion, the finger had reduced sensation. During the time course of occlusion, the restrained finger became darker, displaying signs of mild cyanosis. For some subjects was remarkable, due to the blood pooling in superficial vascular plexus situated in papillary dermis. It has been supported by our observation that mechanical compression of skin tissue produced white patches, as the capillary blood pools were dispersed. The typical cyanotic finger is depicted in Figure 4 on the left side, with the corresponding spectrum acquired from intact and occluded skin, pointing on the obvious difference in hemoglobin maximal absorption spectral region 500-600nm.

Figure 4.

Skin spectra and photo of intact (solid line) and occluded (dotted line) tissue at baseline, in intact finger and occlusion, in occluded finger. The photo of hand was digitally contrasted (25%) to show skin color tones of intact and occluded finger.

In order to hyperspectrally evaluate the reliability of finger occlusion model, an assumption that oxygen is distributed uniformly in occluded tissue region has been made, which is the matter of discussion and controversies in the literature.

The mean baseline value for intact skin saturation acquired by the blood analyzer was 95.13±1.46 %, which resembles arterial blood. Controversial data have been reported in the literature regarding baseline values of skin capillary blood saturation which were recorded with hyperspectral imaging-ranging from 50%-96%^26–31.

The diffusion of gases within relatively small tissue volume depends on many factors such as tissue temperature, gas concentration gradient and tissue mechanical composition, and within 25 minutes should lead to equal distribution of oxygen in skin and underlying soft tissue. Therefore it has been assumed that skin saturation values are represented by saturation of capillary blood taken from the skin microvasculature. The group mean value for HIS derived SaO₂ in intact finger skin was 89.46±8.79%, while substantially decreasing in occluded finger: 25.85±14.00%, displaying small difference between two independent techniques, as seen in Figure 5.

Figure 5.

Bland-Altman plot of agreement between saturation obtained by referent (REF) and hyperspectral method (HIS). From occluded finger during 25^th minute of occlusion (A) and intact finger during baseline (B).

The HIS derived mean saturation value display substantially higher dispersion compared to the referent saturation (standard deviations) indicated on possible differences of skin structure or capillary density across the subjects as the tissue model parameters remained the same.