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Methods of measuring skin blood flow

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1989 Phys. Med. Biol. 34 151

(http://iopscience.iop.org/0031-9155/34/2/001)

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Phys. Med. Biol., 1989, Vol. 34, No 2, 151-175. Printed in the U K

Review article

Methods of measuring skin blood flow

I D Swain? and L J Grant$ Medical Physics Department, Wessex Regional Health Authority + Odstock Hospital, Salisbury $ Royal United Hospital, Bath

This review was completed in September 1988

Contents

1. Introduction 2 . The structure of skin and its blood supply 3. Direct capillary pressure measurement 4. Transcutaneous oxygen measurement (Tcp02) 5 . Radionuclide techniques 6. Temperature

6.1. Radiometric measurements 6.2 Thermography 6.3 Microwave radiometry 6.4 Thermal clearance or conductivity measurements

7. Ultrasound 8. Dermofluorometry 9. Laser Doppler flowmetry

10. Photoplethysmography 11. Capillary microscopy 12. Conclusions

151 152 153 155 157 159 160 161 162 162 163 163 166 168 169 171

1. Introduction

The study of skin blood flow is a comparatively new subject and even old established techniques, such as thermography, have only been used routinely in the clinical environment for a little over 20 years. Today many methods exist for the study of the cutaneous circulation. The number and diversity of these various techniques indicates that absolute determination of flow is not a simple problem and to date no ‘gold standard’ exists. Instead, different techniques measure different aspects of the circula- tion, hence each is applicable under certain conditions and not under others. Three different physiological parameters are used when attempting to describe skin blood flow. These are: (1) physical movement, ( 2 ) heat transport and (3) oxygen content. As the blood flows through the skin surface it transports heat and oxygen to the area, so temperature and oxygen content measurement are relevant parameters. The move- ment of the blood itself to clear away a tracer substance is used in the dermofluorometric and radionuclide techniques, whilst the measurement of this movement is detected using ultrasound and photoplethysmography. Laser Doppler techniques use the move- ment of red blood cells to measure the flow. Only two methods, thermography and

0031-9155/89/020151+2S$02.S0 0 1989 IOP Publishing Ltd 151

152 I D Swain and L J Grant

fluorescein staining, give an indication of regional flow; the remaining methods look at small volumes of tissue of different areas and to different depths. This review surveys the methods of assessment of skin blood flow currently in use, examines the different parameters being measured, and comments on their applicability and problems of interpretation.

Skin blood flow can be considered as comprising both nutritional and non-nutri- tional flow, with changes in the non-nutritional flow acting as a thermoregulatory mechanism. The nutritional component of flow is small when compared with the maximum total flow capacity that is available in the cutaneous circulation. However, it is clinically the most important; if it is not maintained above a given critical level, ischaemia occurs and the skin is no longer viable. Determination of this critical level of flow is obviously of great importance in clinical measurement. Two recently published books give more in-depth information on this subject than can be contained in a brief review (Spence and Sheldon 1985, Tooke and Smaje 1987).

Skin blood flow measurements are used to study clinical conditions which either intrinsically affect skin blood flow or where measurement of skin blood flow is affected by the underlying pathology. The main conditions include: peripheral vascular disease, vasomotor disorders, e.g. Raynaud's phenomenon, skin flap viability following plastic surgery, selection of amputation level and assessing the efficacy of a wide range of treatment regimes.

2. The structure of skin and its blood supply

The skin is the largest organ in the body and provides a boundary between the rest of the body and the outside world. Skin consists of two types of tissue, the dermis which is composed mainly of collagen, a tough fibrous material which gives the skin its tensile strength, and the epidermis which consists of keratinocytes which serve as a protective impervious outer layer.

The epidermis lies above the dermis and itself consists of a number of layers (figure 1). The lowest of these layers is the stratum germinativum, or the basal cell layer, which is only one cell thick and follows the rete pegs of the papillary dermis; this is the most superficial layer in which cells are produced. Pigment-forming cells or melanocytes are also present in the basal layer. Cells migrate from the basal cell level

? l 40-80 pm

~ 300-600pm

1.1-2 mm i

1 700-1KK) pm

Epidermis

Venule Sub papillary dermal Arteriole 1 plexus

Deep reticular dermal plexus

Subcutaneous fo t

Figure 1. Arrangement of blood vessels in the skin.

Methods of measuring skin blood f low 153

through the stratum spinosum, so named due to the spiny looking structures between the cells, tonofibrils, to the stratum granulosum where the cells adopt a more flattened appearance. In the most superficial layers of the stratum granulosum the tonofibrils appear less densely packed and are known as the stratum lucidum. The final layer of the epidermis is the stratum corneum which consists of layers of flattened keratinised cells each 0.5-0.8 pm thick. The number of layers varies with body region, ranging from 15 to 20 on the abdomen and back to several hundred on the palms and soles. The c e k are closely bound forming a barrier to the passage of material in either direction. The complete epidermis is approximately 40-80 pm thick on an adult thigh.

The dermis is considerably thicker than the epidermis, 1-2 mm, and although consisting largely of collagen, also contains cells such as fibroblasts, mast cells and histocytes, and other structures such as hair follicles and sweat or eccrine glands. Unlike the epidermis, the dermis has a very rich blood supply, though no vessel crosses the dermo-epidermal junction. The cutaneous microvasculature consists of a horizontal plexus in the papillary dermis from which the capillary loops in the dermal papillae arise, and a second horizontal plexus that lies at the interface between the dermis and the subcutaneous fat. Arborising vessels join the two plexuses and also provide blood supply to hair follicles and sweat glands via networks of capillaries. The upper plexus consists mainly of post-capillary venules 10-20 pm in diameter, whereas the lower plexus consists of venules and arterioles 50 pm in diameter which have a different ultrastructure to those in the upper plexus. The capillaries found in the skin have an extremely thick wall, 2-3 pm compared with 0.1 pm in the rest of the body.

The capillaries provide the nutritional requirements of the skin, with the effective capillary area being controlled by pre-capillary sphincters. Arterio-venous anas- tomoses also control the amount of blood and hence nutrition reaching the skin by diverting large volumes of blood though low resistance pathways. There has been little evidence to suggest that these anastomosing vessels contribute to the exchange of nutrition and are often known as the non-nutritional part of the microcirculation.

3. Direct capillary pressure measurement

One of the primary techniques for measuring tissue perfusion is the investigation of capillary blood pressure. Whilst this technique, which is usually invasive, is not commonly used in clinical studies, it does provide a useful research tool. Initial studies of direct capillary pressure were made using a mercury manometer with a transparent membrane positioned over the finger nailfold. The capillary loops were observed directly through the membrane. The pressure was increased until capillary blood flow was observed to stop and then slowly reduced until flow returned. This point was taken as capillary blood pressure (Danzer and Hooker 1920). However, subsequent measurement by more direct methods proved this technique to have some errors. The first direct measurements were made by Carrier and Rehberg (1923) who cannulated a single nailfold capillary loop using a handheld glass micropipette. The flow of blood into and out of the pipette was observed with a microscope and the pressure in the pipette altered to stop any flow changes. Subsequently, the technique was much improved by the use of finer pipettes which were mounted in a micromanipulator (Landis 1930).

Modern technology has refined the technique by the use of feedback control to keep the pipette pressure accurately tracking capillary pressure changes (Mahler et al 1979). The pipette is filled with highly conductive 2 M saline. When in the capillary

154 I D Swain and L J Grant

lumen, blood entering the pipette alters the conductivity and this change is detected and used to control the pressure of the saline in the pipette.

This direct measurement of capillary blood pressure has been used in a number of clinical studies to validate other techniques of skin blood flow (Fagrell and Ostergren 1987). Figure 2 shows the relationship between arterial limb capillary pressure and temperature which has been reported by Tooke (1987). Eichna and Bordley (1942) have undertaken the only published study of hypertensive patients with normotensive

a a

2 0 1 I I 1 I I I 29 30 31 32 33 3 4 35 36

Skin temperature P C I

Figure 2. Arterial limb capillary pressure recordings (servonulling technique) from a single capillary during progressive warming and cooling. Courtesy Tooke and Srnaje (1987) and Martinus Nijhoff Publishing.

Figure 3. Capillary Bordley (1942) and

0

0

0

60 - t 8 0 - ! Y

'? 50 -

40 - - 4 3-

30 - * - v

20 - 'I' 9 B 0 0

0 - .f. 09 i 10 -

0 0 - 0

Controls Hypertensives

pressure in patients with hypertension compared with controls. Courtesy Eichna J. Clin. Invest .

and

Methods of measuring skin blood Jow 155

controls (figure 3), which shows that there is only a slight rise in the mean capillary pressure in the study group. Several teams have used the technique to investigate the physiology of other diseases which affect the peripheral circulation (Tooke and Smaje 1987), though there is little routine use of this technique in the clinical environment.

4. Transcutaneous oxygen measurement (Tcp02)

The major role of the skin circulation is to supply nutrients to the tissue to keep those tissues alive. Therefore it would appear that measurement of the skin oxygen content would be an ideal indicator of skin viability, if not actual skin blood flow. However, as skin is not homogeneous the amount of oxygen present varies from a minimum value in the tissue cells to a maximum value in the arterial circulation. Due to these difficulties few attempts have been made to use these measurements with patients (Montgomery and Horwitz 1950, Spence and Walker 1976, Spence et al 1985).

In order to overcome the problems of measuring tissue oxygen content by a microelectrode, transcutaneous oxygen measurements have often been used as an indicator of tissue viability (Baumbach 1985).

Transcutaneous measurement of oxygen tension was first developed as a non- invasive method of continuously monitoring the arterial oxygen content in neonates (Huch et a1 1973) and today this technique is very widely used in special care baby units. Evans and Naylor (1967) showed the PO, on the surface of normal skin is practically zero, and that the pOz rose following vasodilation either by drugs or by radiation. Huch er a1 (1973) described a skin-mounted oxygen sensor with a tem- perature-controlled heater included. They found that when the skin was heated to 42-45°C vasodilation occurs within the capillary bed causing hyperperfusion under the electrode. The electrode used is a Clark electrode (Clark 1956) and consists of a polarographic cell covered with an oxygen permeable membrane. Molecular oxygen diffusing from the warmed capillaries is reduced at the cathode by the application of a potential of 400-850 mV between a noble metal cathode and a reference anode (Rozkovec and Rithalia 1980). As oxygen is consumed at the cathode it will be replaced by oxygen diffusing through the skin. When the reduction process is limited by the rate of diffusion the measured current will be directly proportional to the PO, in the underlying structures.

The structure of skin is shown in figure 1 and for the purposes of TcpOz measurement can be considered to consist of three layers, the capillary dermis from which oxygen diffuses, the basal layer in which oxygen is consumed, and the stratum corneum, the outermost layers of the skin in which oxygen consumption is zero. A number of papers have dealt in detail with the pOz profile through these various regions under a variety of differing conditions (Huch er a1 1979, Grossmann and Lubbers 1983). Therefore, the measured TcpO, value on adult skin will not only be a reflection of the arterial p 0 2 but is also dependent on skin thickness, tissue/skin metabolism and the conditions of the capillary bed (Shakespeare and Swain 1985). In addition, electrode characteris- tics such as oxygen consumption, response time and skin heating profile affect measured values. Different electrodes placed side by side can give markedly different TcpOz values, particularly in skin ischaemia where electrode oxygen consumption might be a significant proportion of oxygen availability (Spence et ul 1985).

The depth from which the oxygen diffuses is also not known and will depend upon the characteristic of the heater in the probe. Shakespeare and Swain (1985) showed that the measured TcpO, level fell when the skin under the electrode had been treated

156 I D Swain and L J Grant

40 r

g ::: 0 10 2 0 30 4 0 5 0 60 70 80 90

T C ~ O ? (mm Hg l

Figure 4. Graph showing results of Dowd TcpO, measurements. Courtesy Dowd (1982).

30 r

l "l L

0 60 120 180 240 300 Tlme a f t e r release of cuff (5)

Figure S. Forearm TcpO, i S D after a 10 min period of ischaemia by sphygmomanometer cuff at 200 mm Hg. SD values for minimal retinopathy group have been omitted for clarity.-, Normal; Diabetic: - - -, nil/minimal retinopathy; . . . proliferative retinopathy. Courtesy Newson and Rolfe (1987) and Martinus Nijhoff Publishing.

Methods of measuring skin blood flow 157

with rubefacients. Observation by capillary microscopy showed an absence of patent capillaries in the area but an increase in the size of deeper anastomosing vessels. The fact that the TcpO, was reduced, but not to zero, indicates that oxygen was diffusing from deeper structures in the skin.

The interpretation of TcpO, values obtained from ischaemic skin is especially difficult and it is unreasonable to assume that such values reflect the underlying tissue PO,. Low or even zero readings are common, despite evidence of capillary filling and positive blood flow indicated by different techniques (Spence 1985). It has been proposed (Wyass er a1 1981) that TcpO, values are related to the local microvascular transmural pressures which increase in proportion to the hydrostatic pressure changes in the ischaemic foot.

However, despite the difficulties noted above, TcpO, has been used successfully for assessing the condition of the peripheral circulation in the assessment of ischaemia prior to amputation (Dowd et a1 1983), and in the effect of prolonged pressure loading on the skin. Figure 4 shows the results of TcpO, studies in patients with claudication, ischaemia and gangrene compared with normals (Dowd 1982); whilst figure 5 shows Newson and Rolfe's (1987) study of the effect of total occlusion of blood flow on Tcp02 values over a 5 min period. Bader and Gant (1985) and Spence er a1 (1985) have proposed that dynamic changes in oxygen tension, induced by inhalation of oxygen, might prove to be a better indicator of tissue viability. Similarly, simultaneous measurement of p 0 , and p C 0 , would give additional information on skin metabolism. This is now possible with the new generation of combined sensors but it is too recent a development for its value to be established.

5. Radionuclide techniques

Subcutaneous injections of various substances labelled with radioactive isotopes are used to evaluate skin blood flow. The activity in the area of the injection is measured and this is related to the rate of clearance of the isotope by the local blood supply. The first use of radioactive isotope techniques for the investigation of skin blood flow used radioactive krypton, %r (Smith and Morales 1944). This was closely followed by another group who used sodium, 24Na (Smith and Quimby 1945). Both these studies used intravenous administration and provided primarily a qualitative assessment of skin blood flow. The first quantitative study used 0.2 MBq of 24NaC1 in a subdermal injection to demonstrate local washout due to blood flow (Kety 1949). Calculation of the tissue blood flow is possible by estimating the half-life of the tracer ( from the washout curve and applying the formula

Q = m1 (100 g tissue)" min" 100 In 2 A

( t0 .S)

where A is the tissue/blood partition coefficient. Many tracers have been used for skin blood flow measurement such as '33Xe, 85Kr,

*,Na, 24Na, 99T~m, '''I or '311-4-iodoantipyrine (Spence er a1 1985). Clearly each of these isotopes has a range of advantages and disadvantages. Xenon clearance has been the method of choice for many years and is the technique which has perhaps come nearest to a 'gold standard', following work by Sejrsen (1969). References to studies using radionuclide techniques in the literature are numerous and constitute primarily clinical studies.

158 I D Swain and L J Grant

The main problem in determining which substance to use as a tracer is the degree of diffusibility after a subdermal injection. Freely diffusible indicators which have lipophilic molecules are often preferred to restricted diffusible molecules which have hydrophilic molecules. However, the validity of the study will depend upon the level of subdermal fat which can affect the washout curves of the lipophilic tracers. Finney er a1 (1985) used these objections, combined with the ready availability of technetium in nuclear medicine departments, as criteria for using 99Tc in their study. Currently,

I-Ciodoantipyrine is receiving much attention and support since it is much less fat soluble than xenon and is freely diffusible (McCollum et a1 1985). Problems have existed with this substance due to the presence of free unbound iodide. These have been overcome recently by the introduction of a new preparation technique which can be undertaken in a hospital radiopharmacy (Forrester et a1 1980).

Irrespective of which isotope is used all studies involve a subdermal injection of the tracer and the use of a suitable detector to measure the washout at that particular point. This technique does have the considerable disadvantage that it is very much a point measurement and does not necessarily bear any relationship to regional blood flow. The other point measurement techniques described in this paper do, in general, just take a point as part of a whole regional consideration. By studying the effect on a local subdermal injection there is the possibility that a poor site has been selected and therefore inaccurate results may be obtained. The accuracy of the technique will, of course, improve with multiple site measurements. This can, however, lead to quite a lengthy study.

The half-life of the tracer element is a factor which affects the study and must be taken into account when calculating the washout curve. These times vary between the 6 h of 99Tcm to 59 days for 1251. The biological half-life is also a factor since the

125

1000 -

800 - 133 X e b F :l 01

i 400 01 '"il-41Ai'(EF - 2 . 2 ) - - m Y

200 -

I I I I l 0 S 10 1s 2 0

Tlme i m i n l

Figure 6. Simultaneous semilogarithmic clearances of '"Xe and '*'1-4-IAP following intradermal injection (in a mixed bolus) into a critically ischaemic skin site. At these low flow rates the difference in washout slopes between the two tracers is clinically significant. Courtesy Spence er al 1987 and Martinus Nijhoff Publishing.

Methods of measuring skin blood flow 159

effective half-life (Teff) is obtained from the relationship

1 1 1 Teff T p h y s Thiol

- +-

where T p h y s is the radioactive half-life and Thiol is the biological half-life. Information on the biological half-life is not readily available and, in any case,

depends upon the area in which the tracer is administered. Thus the lipophilic and hydrophilic molecules can have a far more significant effect on the effective half-life than the radioactive half-life. Spence et a1 (1987) have shown the different count rates which are obtained over a 20 min period with '33Xe and '251-4-iodoantipyrine (figure 6). Therefore care needs to be taken to assess the skin tissue type in the area to be studied in order to use the appropriate tracer. This can, of course, be further aggravated by the biological variation between patients.

6. Temperature

Like several of the other techniques available temperature can be used as a local or regional indicator of skin blood flow. Readings can be obtained by point contact thermometry or radiometry, whereas thermographic imaging gives an indication of regional blood flow. Thermal clearance or conductivity techniques are also extremely useful as indicators of local skin flow.

One of the problems in using temperature as a measurement of skin blood flow is the difficulty in defining the different contributions of heat flux from specific segments of the dermal vasculature, although undoubtedly the capillaries and venules act as the greatest heat exchangers. The ability of the venules to act as a reservoir for large volumes of slow-moving blood has the most significant impact on heat exchange in the cutaneous layers of the skin (Spence et al 1985). Arterio-venous anastomoses will modify the heat exchange because of their ability to short-circuit large volumes of blood into the superficial venous plexus.

Although very few people have claimed to be able to quantify actual values of blood flow, skin surface temperature is commonly used as a clinical indication of peripheral vascular disease, one of the main reasons for wanting to measure skin blood flow (Henderson and Hackett 1978, Spence et al 1981).

The skin temperature is, of course, affected by both internal and external factors (Houdas and Ring 1982). The skin is heated by tissue conduction, the heat being produced by underlying organs and tissues and its contribution to the overall skin temperature will thus depend on the thermal conductivity of the subcutaneous struc- tures. In addition, blood convection warms the skin by transfer of heat from the core and it is this factor which plays the major part in determining skin temperature. The skin will, of course, constantly lose heat into the environment by all three methods: radiation, convection and conduction. It is most important therefore that the conditions under which skin temperature is measured are very closely controlled and defined.

There are three factors which affect skin temperature: blood perfusion, tissue ther- mal conductivity and metabolic heat generation. Love (1980) has shown by mathem- atical modelling techniques that it is blood perfusion which dominates the overall effect, and has claimed that quantitative measurements of blood flow are possible with tightly controlled conditions and a rigorous analysis. This hypothesis has, however, been challenged by Brown et a1 (1980) who in the analysis of the thermal clearance technique

160 I D Swain and L J Grant

maintained that the thermal flux due to blood flow is less than that due to the tissue thermal conductivity. Houdas and Ring (1982) have shown the relationship between change in blood flow using venous occlusion plethysmography and skin temperature is non-linear (figure 7) .

L! 1 1 I I

0 l0 20 30 40

Skin temperature ('C )

Figure 7. Blood flow in the hand (plethysmography) related to water bath temperature. Courtesy Houdas and Ring (1982) and Plenum Press.

Adams et al( l980) have described an invasive technique for accurate quantification of regional blood flow. In this technique they used thermocouples carefully implanted subdermally, which whilst being of value in animal studies into other techniques, is clearly unacceptable for normal clinical measurements.

6.1. Radiometric measurements

According to Wein's law, the frequency at which the maximum energy is dissipated depends on the temperature of the emitting object. For example, radiation from the human body at 37°C is maximal at a wavelength of 10 pm. This is in the infrared region. However, infrared is easily absorbed by living tissue, and hence only radiation emitted by the skin surface can be measured by this method (Houdas and Ring 1982).

All living bodies emit electromagnetic radiation with the total energy emitted depending on the absolute temperature and emissivity of the body. Although the amount of energy emitted from the human body is very small it is detectable. This radiation covers a large range of wavelengths and thus infrared radiometers can be used for non-contact measurement of skin surface temperature. Hardy (1934) has shown that skin behaves as an efficient emitter of heat with an emissivity close to that of a black body radiator (i.e. nearly 1.0). His experiments showed an optimal emission from skin at 30 "C at around 8-10 pm (figure 8). Most clinical infrared radiometers have a sensitivity matched to the skin over the range 2-25 km.

Methods of measuring skin blood flow 161

2 S 10

Wavelength ( k m 1

Figure 8. Emissivity of skin related to wavelength. Courtesy Hardy (1934) and J. Clin. Incest.

Care has to be taken when using these instruments since the angle of the sensor head to the skin and the distance from the skin are important, although angles less than 45" seldom cause problems. The instrument displays the mean temperature over the area recorded and this, although small, depends on the individual sensor and its focusing arrangements. This has to be considered when measuring areas which will have temperature discontinuities, e.g. near major surface blood vessels.

Most accurate radiometers use a bolometric principle (Ring 1986). A thermocouple Wheatstone bridge circuit is used with one thermocouple referenced to a heated chopper. The detector unit contains the other thermocouple mounted in a vacuum tube.

6.2. Thermography

Thermography uses the same principle as the radiometric measurement. The energy flux emitted by the skin is given by Stefan-Boltzmann's law

where 6, is the Stefan-Boltzmann constant and T, is the skin surface temperature. This radiation is detected and converted into an image of the regional distribution of temperature.

Traditionally detectors used indium antimonide which is sensitive in the range 2-5 pm. In order to obtain sufficient signal-to-noise ratio the detectors are cooled using liquid nitrogen. More recently mercury cadmium telluride detectors have been introduced with a maximum sensitivity at 12-15 pm. It will be noticed that the response spectrum for these two detector materials and typical radiometers are different. Care must be taken therefore to interpret the results when using more than one technique.

In any measurement of surface temperature it is important to let the patient's skin temperature stabilise, without clothing, for at least 15 min. The temperature of the room relative to thermal neutrality of the body is important to avoid any unwanted heat gain or loss and an experienced thermographer will be able to enhance his images by judicious use of room temperature (Houdas and Ring 1982).

162 I D Swain and L J Grant

Two new techniques have recently been introduced as the result of new detector technology. Both were originally developed for military or industrial applications, dispense with the need for liquid nitrogen cooling, and have outputs which are compatible with conventional television technology and frame rates.

The first technique uses an array of indium antimonide or cadmium mercury telluride detectors which are arranged in eight strips, the strips constituting a serial array. This is known as the SPRITE (signal processing in the element) detector (Elliott 1981). The sensitive element of a SPRITE detector is elongated in the direction of scan and biased in such a way that the carrier drift velocity exactly matches the scan velocity. The strip is then mechanically scanned and the signal extracted with only three connections and one preamplifier.

The second technique uses conventional television vidicon tubes and replaces the sensor surface with a pyroelectric material 20 mm in diameter and 30 pm thick. Pyroelectric materials are asymmetrical crystalline materials having an internal electric dipole moment which is temperature sensitive (Burgess 1984). The faceplate must, of course, transmit infrared and germanium is used along with a germanium lens. The infrared radiation causes localised changes of target temperature which are converted into corresponding surface charges on the rear face of the target. This is displayed in the conventional way by the raster-scanned electron beam.

6.3. Microwave radiometry

Because infrared thermography (2-25 pm) is only able to measure surface temperature the microwave frequencies of 1-10 GHz are now being investigated. The emitted energy is very low, 10-14 kW, which is about lo7 times lower than that at 10 pm. Its advantage is that human tissue is partially transparent to microwaves, to a depth of 1 or 2 cm. Therefore, the technique has the ability to detect the temperature of subcutaneous tissues. Manson et a1 (1985) have recently used microwave thermography to assess peripheral vascular disease and found that it was more clinically useful than infrared thermography.

6.4. Thermal clearance or conductivity measurements

Thermal clearance is a technique which, it is claimed, gives a very good approximation to skin blood flow using a contact measurement technique in conjunction with a thermal stimulus (van de Staak 1966, van de Staak et a1 1968, Manson er al 1985). The probe is usually an array of thermocouples around a circular heater which is used to raise the skin temperature by several degrees. The mean difference in temperature with and without the heater operating is recorded. As heat is conducted away by the blood vessels in the area, the greater the flow the lower the temperature difference will be.

Once again, care needs to be taken when using this technique. The heater and thermocouples need to be separated from each other (at least 1 cm) due to convective heat transport in the area, and the effects of subcutaneous tissue need to be evaluated. van de Staak et a1 (1968) developed a phantom technique for validating this method whilst Britton et a1 (1984) have undertaken mathematical modelling to show the importance of fat and blood flow in affecting thermal conductivity. Brown et a1 (1980) have also indicated the importance of blood vessels within a few millimetres of the probe in extracting heat from the immediate area of the sensor and thus reducing the temperature difference.

Methods of measuring skin blood jlow 163

By altering the geometry of the probe it is possible to obtain information from different depths into the skin and subcutaneous tissue (Britton et al 1984). Holti and Mitchell (1978) have performed experiments with acrylic sheets which suggest that the penetration effect is of the order of a few millimetres. Acrylic sheet has a conductivity which is close to that of bloodless skin. Brown and colleagues have established, with the type of probe they used, that thermal flux due to conductivity in normal skin may be 38 mW, whereas that due to blood flow was only 7 mW (Brown et al 1980).

7. Ultrasound

Of the techniques currently available for measuring skin blood flow ultrasound is the newest and is being investigated in only a few centres. Most of the published work to date has been generated by Payne et al (1985), who indicate that the technique holds promise for being able to select the depth from which the measurement is to be taken.

Doppler ultrasound has, of course, been used for many years to investigate blood flow, or more accurately, velocity, in the major arteries (Atkinson and Woodcock 1982). However, it is normally used with transducers which operate at frequencies up to 10 MHz. This is much too long a wavelength to be able to make velocity measurements in the epidermis or dermis which are only 100-500 pm thick. In order to undertake skin blood flow measurements Frew and Giblin (1985) showed that the frequency needs to be between 70 and 90 MHz. By a mixture of experimental and computational techniques they have shown that the acoustic attenuation increases with frequency to the power 1.2 up to 100 MHz and with frequency to the power of 2 above 100 MHz. Their measurements were taken up to 60 MHz by using 6 mm diameter lithium niobate disc transducers which had been polished to increase their operating frequencies to 60 MHz. Faddoul (1985) has used frequencies varying between 15 and 50 MHz and ultrasound fields which are focused as well as flat ones with a diameter of 1 cm. Payne et a1 (1985) have reported that using Doppler ultrasound skin blood flow measurements can be taken from a predetermined layer within the skin. Their technique is to use the tone burst signal in an A-scan mode to find the various interfaces from the received echoes. The receiver gate control is then set to collect data just from this region.

Apart from the high frequencies involved in these measurements, the other main problem is the low Doppler shift frequencies arising from the low flow speeds encoun- tered in the skin blood vessels. Flow rates which are as low as 0.1 mm S" will give rise to only a few hertz of Doppler shift frequency. This gives extreme problems with signal-to-noise ratio. Fourier transform techniques are used to extract the necessary flow information from the Doppler shift frequency. With their equipment Payne and his colleagues managed to measure frequency shift in the range 0-20 Hz which took 25 S to analyse.

Thus although Doppler ultrasound techniques hold promise, the technical problems in obtaining satisfactory results remain considerable.

8. Dermofluorometry

The use of sodium fluorescein in conjunction with ultraviolet light is a technique which has been known for a number of years (Lange and Boyd 1942) and with thermography, is primarily aimed at regional blood flow investigation. The majority of research in

164 I D Swain and L J Grant

this field has been undertaken as a result of a need to predict the success, or otherwise, of skin flap surgery (McGraw et a1 1977, Silverman er a1 1980, Myers and Donovan 1985). As with all techniques an increasing desire for quantification has led’to the development of instrumentation. However, the technique is still extensively used with only qualitative assessment of skin blood flow.

Tissue fluorescence is taken to be an indication of the extent of nutritive blood flow because following an intravenous injection of sodium fluorescein the dye accumu- lates in the extracellular fluid by passage across capillary walls (Spence et a1 1985).

Although the technique has been used since 1942 for skin blood flow measurements, it was Ehrlich (1882) who originally used the dye to investigate the secretions of the aqueous humour in the rabbit’s eye, its flow through the eye and its absorption. In those days fluorescein (resorcinol phthalein) was used in an alkaline solution of sodium bicarbonate since it has about 28% greater fluorescence than sodium fluorescein (Lange and Boyd 1943).

When sodium fluorescein is injected into the venous circulation and viewed with blue or UVA light in the band 450-500 nm there is a green-yellow glow in the range of wavelengths 530-660 nm. The light excites the electrons of the fluorescein molecules and briefly raises them to a higher energy level. Photons are released as the electrons return to their ground state which results in the generation of the fluorescence. This can give quite readily identifiable results when there are non-perfused areas which fail to exhibit the characteristic glow. The technique can therefore be routinely used in operating theatres to establish a first line assessment of the flap viability (Singer er a1 1978, Myers and Donovan 1985). However, the more difficult task of assessing relative perfusion cannot be accomplished without fluorometry instrumentation, the eye being a poor interpreter of the relative intensity of the fluorescein ‘glow’.

Thus the need arose to turn the simple observation of areas of perfusion into a method which would quantify the blood flow at that point. The original work on this technique was undertaken by Lange and Krewer (1943) who developed the dermo- fluorometer. Their instrument consisted of an incandescent light source, a blue excita- tion filter to selectively transmit the light to the tissues and a green emission filter to screen out reflected blue light while permitting passage ofthe yellow-green fluorescence. A phototube converted the photons to a current which was read on a galvanometer. It was used for many years but there was difficulty with patient acceptability because of its cumbersome size.

The technique has, however, been subsequently developed by Silverman et a1 (1980) using more modern technology and is now known as the fibreoptic dermofluorometer. This is commercially available as the Diversatronics 2000 Perfusion Fluorometer (Diversatronics Inc, 620 Parkway, Broomall, PA 19008, USA). Silverman’s technique uses an incandescent light source transmitted through an excitation filter which selec- tively transmits wavelengths between 450 and 500 nm within the range of maximal excitation of fluorescein in vivo (Delori et a1 1978). This light is then passed down a fibreoptic bundle to the probe tip which is in contact with the tissue. Fluorescent emission from the tissue under the probe and reflected excitation light pass back along other fibreoptic pathways and are filtered to transmit only wavelengths in the region 530-660 nm which are then detected by a photomultiplier tube. This output signal is converted to a value which is called dye fluorescence ( D F ) units. The instrument is linear with dye concentrations in the range 1.25 x 10”o-l X g ml” in vitro. The value of the DF unit can then be normalised by comparison with an area of normally perfused tissue, the ratio being known as the dermofluorometric viability index ( D V I ) .

Methods of measuring skin blood f low 165

After injection of the dye the concentration peaks and reaches a plateau over a period of 10-20 min. However, readings need to be maintained for up to 30-40 min as the dye gradually perfuses from the pericapillary tissue to tissues more distant from capillary flow. If all dystrophic tissue needs to be excluded in the study then the shorter time period will produce accurate results. However, the time of fluorescein delivery, which is variable, is a parameter which may help to predict more precisely the degree of ischaemic change in areas destined to become dystrophic.

Leopold et a1 (1985) have recently shown that several other factors can affect the readings obtained from these instruments. These are (1) skin pigmentation, (2) erythema and (3) the distance between the measuring probe and the interstitial fluid space. This confirms work undertaken by Brousseau er a1 (1983) where they found marked differences between readings taken on Negroid and Caucasian skin.

Gratti et a1 (1983) used the technique to investigate burn wounds and found that if studies were extended to 60 min they could accurately differentiate between full and partial thickness burns early in the postburn period. They could not, however, differentiate between superficial and deep partial-thickness burns. Silverman et a1 (1981) have shown significant differences in washout patterns in tissue with different viability (figure 9). This makes the technique extremely useful in predicting the outcome of surgical procedures or tissue trauma.

Bongard et a1 (1984) have studied the correlation between blood flow and fluores- cence and found that the intensity of tissue fluorescence is linearly related to blood flow and that conclusions regarding perfusion may be drawn from relative fluorescence at any time between 20 and 60 min following a bolus injection. In this study on the

2 . 2 -

2.1 -

2 . 0 -

1.9 -

1 . 8 -

1 . 6 -

1 . 5 -

2 5 1 4 -

2 0 .I 1 3 0 5 0 100 150 200 250

Time (mm) Figure 9. Fluorescein elimination in a representative experimental animal. Three distinct ‘washout patterns’ were discernible on day 1. These provided three straight lines when ‘log DF’ against time was plotted. Rapid elimination was observed in the tissue destined to remain viable. On the other hand, no elimination was observed in the tissue destined to become dystrophic. Courtesy Silverman er ai (1981) and Surgery.

166 l D Swain and L J Granl

hind legs of rabbits they controlled the blood flow into the region of measurement with a distal aortic sling and then used an electromagnetic flowmeter to continuously measure the blood flow. They also concluded that the passage of fluorescein from the blood stream into the interstitium, where it becomes available for analysis, is dependent on a rate-limiting diffusion process and the amount of accumulated fluorescein that must return to the intravascular compartment for elimination.

9. Laser Doppler flowmetry

The coherent light produced by lasers may be utilised to measure Doppler frequency shifts when reflected from moving red blood cells. The use of the laser Doppler technique to study the circulation was first described by Stern (1975). The surface of the skin was illuminated by a 15 mW He-Ne laser. The reflected portion of the incident beam was found to be spectrally broadened by the Doppler effect, primarily due to moving red cells in the superficial blood vessels, although it will be affected by all movement within the hemisphere which is illuminated (Tenland 1982).

The first portable clinical instrument was developed by Holloway and Watkins (Holloway and Watkins 1977, Watkins and Holloway 1978) although this had some practical limitations due to a poor signal-to-noise ratio. An improved laser Doppler flowmeter (LDF) was reported by Nilsson er a1 (1980) which uses a dual-channel detector. This allows differential amplification of the reflected signal removing the common-mode signal of laser noise.

The scattering of light in tissue is complex. As the red blood cell is an irregular structure of an order of magnitude larger than the wavelength of the laser light, with a refractive index near to that of tissue, scattering is predominantly in the forward direction according to the Rayleigh-Debye theory, with less than 0.lOi0 backscattered by moving red cells (Bonner and Nossal 1981). This scattering of light in the forward direction results in a significantly lower average frequency shift of the light compared with backscattering. In practice the majority of photons are scattered from stationary tissue. However, some are scattered by moving blood cells and undergo a frequency shift, as well as being scattered by stationary tissue. In tissue with a high concentration of red cells light may be scattered by more than one moving cell. With multiple scattering it is practically impossible to theoretically calculate the Doppler spectrum (Stern 1975).

However, light being scattered from moving red blood cells will undergo a frequency shift according to the Doppler equation

f = (2ufocos O ) / c

where u is the velocity of the red blood cell, f o is the frequency of incident laser light, c is the speed of light in tissue and 8 is the angle between the incident ray of light and the direction of motion of the red blood cell.

The absolute value of the Doppler shift can only be calculated if O is known. However, in practice where each photon undergoes multiple scattering from many red blood cells O may vary between 0 and 180" for each scattering event. Therefore, the measured frequency shift is derived from an average value of 0, the magnitude of which is unknown. The velocity of red blood cells in cutaneous circulation is of the order of a few mm S", and correspondingly produces a Doppler shift of several kHz. This is extremely small compared with the lasing frequency of 5 X lOI4 Hz. Therefore,

Methods of measuring skin blood flow 167

optical heterodyning (i.e. producing a beat frequency) is needed to record the Doppler shifts (Fairs et a1 1985).

The most common approach to analysis of the signal is to use a weighted estimate of the power spectrum. The weighting function varies between different flowmeters, some using a weighting term of the frequency (Nilsson 1980), and others a weighting term of the square of the frequency (Stern 1975). Both methods have been found to correlate well with other methods of skin blood flow measurement (Watkins and Holloway 1978).

The output signal is related to the flux of red cells, i.e. the number of cells times their velocity. This relationship is valid irrespective of how the product of red cell density and velocity is obtained. However, its validity has been questioned in very highly perfused tissue (Tenland 1982).

Laser Doppler flowmetry gives a measure of the integrated motion of red cells in many small vessels within the measuring volume, irrespective of the direction of cell movement (Salerud et a1 1982). As skin is not homogeneous the measured value is critically dependent on the location of the probe. The method is therefore best if kept at a constant location to observe changes at a given site. Ryman (1987) has shown marked changes in vascular response to a pin prick which is significantly different in normal and diabetic subjects (figure 10). The main disadvantages of the method are that it is non-linear at high concentrations of blood cells and that it is sensitive to movement artifacts, although the latest instruments are considerably better in this respect. In addition the effect of dermal configuration, thickness of the epidermis and haemoglobin content of the blood is not known. Therefore, although this method is easy to use, gives real time output and is able to measure minute blood flow, it is difficult to perform true quantitative measurements (Tenland 1982). In studies with large numbers of patients it is possible to compare changes in blood flow in different groups in response to changes in posture (Ryman 1987). Here the numbers studied

Oso r Needle orick

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1 1 "J _ _ _ _ _ _ L _ _ _ _ _ l _ _ _ _ _ J _ _ _ _ _ _ _ J"-+'-J

0 5 10 15 30 60 90 120 180 21,

Time min h

Figure 10. Vascular response to pin prick in a normal individual (dotted curve) and a diabetic subject (full curve). Courtesy Ryman (1987) and Martinus Nijhoff Publishing.

168 I D Swain and L J Grant

,- p < 0.002 T

p< 0.02 1- N S 1

100 1 t

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O L Diobetic Oiobetic Normal neuropothy controls controls

Figure 11. Flow on dependency expressed as a percentage of preceding rest flow in diabetics with and without retinopathy and matched controls. Courtesy Ryman (1987) and Martinus Nijhoff Publishing.

allow statistically significant differences in the mean value to be determined (figure 11). However, the wide spread of recorded values means that individual comparison from one subject to another is not clinically significant.

10. Photoplethysmography

Photoplethysmography is a technique which uses the transmission or reflection of light from the capillary bed to provide information on the local skin perfusion. Obviously the first method is only suitable for assessing blood flow in certain areas such as earlobes, axillary fold, fingers, pedicle flaps etc. If this was the only mode in which this technique could be used, then its clinical applications would be rather limited. However, when light is transmitted down into the skin part of it is backscattered and may be detected. The amount of light incident on the detector will depend on three factors: (1) the attenuation of the tissue, assumed constant; (2) the amount of blood in the vessels of the skin, the DC component or total blood volume; (3) the variation of the volume of blood with time, the AC component or blood pulse volume.

The fact that the signal detected by the photoplethysmography transducer consists of two components was first determined by Hertzman and Speakman (1937) in the course of one of the first papers published on the application of photoplethysmography. A history of this technique and its applications is given by Challoner (1979).

Early workers in the field were handicapped by the size of both light sources and detectors. Today, however, due to advances in semiconductor technology both trans- mitter and detector can be mounted together in a small probe and applied to any part of the body. A great amount of work has been undertaken into the optical processes

Methods of measuring skin blood $ow 169

involved in the production of the signal, although the exact origin and meaning of the signal has not been resolved. To quote Fairs and colleagues (1985) ‘there is a popular belief that flow is being measured’.

Light leaving the source enters the skin and is scattered, reflected, refracted and absorbed and finally reaches the detector. As the volume of blood in the skin changes there will be more or less light detected. If it is assumed that the change in absorption of the light signal is due to absorption by the blood, then the more blood present in the area of investigation, the less the amount of light received by the detector (Weinman 1967). This simple model was developed by D’Agrosa and Hertzmann (1967), Chal- loner (1979) and de Trafford and Lafferty (1984) who showed that the orientation of the erythrocytes also affects the signal recorded at the detector.

The DC component is a measure of the total blood volume, not flow, and as such is affected by pooling due to venous obstruction. In practice, it appears (Challoner 1979) that larger errors may be associated with the measurement of total blood flow due to such problems as non-linearity of the detector, ambient light and the change in optical absorption characteristics of blood depending on its oxygen content.

The detector consists of a single or multiple light source and photodetector encased in a material to prevent extraneous light from reaching the detector. The wavelength of the light is of fundamental importance since longer wavelengths penetrate deeper. It can be considered beneficial therefore to use a light source of narrow bandwidth between 800 and 1200 nm (Tahmoush 1986), corresponding to a penetration depth of 1.2-2 nm (Anderson 1981). Heating effects can be minimised by using optical fibres to illuminate the skin. Two factors determine the choice of detector; first, the pulsatile component is only 0.1% of the DC component, and second it must ideally be matched to the sensor.

Photoplethysmography is non-invasive and can be used to give an indication of the pulsatile flow and the total blood content in the peripheral circulation. However, it cannot differentiate between nutritional capillary flow and flow through the deeper anastomosing vessels. Although the exact methods in which the changes in blood flow modulate the light signal are not fully understood, useful information on the cutaneous circulation can be determined by careful technique.

1 1. Capillary microscopy

Capillary microscopy follows the old biologists’ adage of ‘when in doubt look at it’. These observations can fall into two categories: first to observe the state of the capillary bed, and second to measure the velocity of red blood cells in those capillaries. The advantage of this technique is that direct observations can be made and any abnormality seen. The limitation of this technique is that as the majority of capillaries run perpen- dicular to the surface of the skin they are difficult to observe, making the measurement of red blood cell velocities virtually impossible. However, this technique can readily be used for observation of nail fold capillaries which run parallel to the skin surface, and also capillaries in areas of abnormal vasculature such as port wine stains, hyper- trophic scars and around wound sites.

A stereo microscope is used with a long working-length objective, typically 20x magnification. Illumination is by blue-green light, ideally 540 nm (e.g. a 50 W mercury vapour lamp), the maximum adsorption frequency for haemoglobin, transmitted through a fibreoptic cable to minimise local heating. Liquid paraffin or clear nail varnish is then applied to the surface of the skin to prevent light scatter. The area of

170 I D Swain and L J Grant

Table 1.

Investigator Velocity (mm S - ' Conditions ("C)

Fagrell mean 0.65 * 0.3 Skin temp. 30.452.3 Bollinger artery 0.84* 0.53 Room temp. 22-25

Tooke mean 0.46 * 0.24 Room temp. 22-24 venous 0.47 * 0.29

Classification of skin capillary stages according to Fagrell

Risk of skin necrosis Classification system

Seven stages Three stages

None 1: 1:

small dot - or comma shaped

slightly dilated

Almost none widely dilated, "micropools" l A indistinct capillaries

Sfoderate high 4 capillary haemorrhages 1 s

Immediate blood filled capillaries 5 pronounced reduction of

1, no blood filled capillaries i c Figure 12. Classification of skin capillary stages according to Fagrell. Courtesy Ostergren and Fagrell (1987) and Martinus Nijhoff Publishing.

150 1 .

. ... . .. .. . 50 - .. .

0 . ... . .. 0

a . ..m ...

2 S - .......................... M R ..... .. S. ........ .R ..... . ... . . . .. . ._ ... ...... .. ._ 0 0 . . M . . . . .. M a.. 0. ooooo

0 000 0 0 -

0 1 2 3 4 5 6 Capillary stage

Figure 13. The relationship between the systolic blood pressure of the big toe (SBP) and the worst capillary stage found in the skin of the investigated toes and forefoot. 0, patients devloping skin necrosis; 0, patients not developing skin necrosis. Courtesy Ostergren and Fagrell (1987) and Martinus Nijhoff Publishing.

Methods of measuring skin blood flow 171

skin under investigation has to remain stationary relative to the microscope if photo- graphs are to be taken, especially at high magnification where light levels are low and depths of field small.

Using a video system overcomes a number of these problems and enables blood cell velocities to be measured (Bollinger 1974, Fagrell et a1 1977, Tooke et a1 1983) by observing the distance a given erythrocyte moves between successive video frames. This can be determined by a video cursor. The values obtained for red cell velocities are shown in table 1.

In addition to this, Bollinger noted a slower variation in flow with a 6-10 S period. Direct observation of skin in other parts of the body can also yield meaningful results. Ostergren and Fagrell (1987) have described a method of classification of skin capillary stages and the morphological changes which take place in patients with severe arteriosclerosis (figure 12). These stages were then compared with the systolic blood pressure of the big toe (figure 13). Shakespeare and Swain (1985) noted that the state of the vascular bed, modified by rubefacients, could be observed, with the relative proportions of capillaries and anastomosing vessels corresponding to the measured Tcp02 values. Other areas in which this technique could yield useful results are in the study of hypertrophic scars, port wine stains and in the borders of chronic open ulcers.

12. Conclusions

It is apparent that at present there is not a ‘goid standard’ method of measuring skin blood flow with which other methods can be compared, as different methods sample different parameters.

Before choosing a given method it is necessary to ascertain what clinical information is required and how that information is to be used in the treatment of the patient. Each method measures slightly different aspects of peripheral circulation and has associated advantages and disadvantages. It is therefore essential that before adopting a given method for clinical use, the user fully understands what that method measures, in relation to the structure and function of the skin. Table 2 gives a summary of each technique, what they measure, their ease of use and their limitations.

The difficulty in fully understanding exactly what each method measures, combined with the complex nature of the skin blood supply, has led to unreliability in basing clinical decisions on measured values. Hence each method has undergone periods of popularity and periods of vilification and disuse. These normally follow a pattern of great enthusiasm when a technique is developed, followed by a period of almost total disuse when its limitations are exposed, before finally finding its own ‘ecological niche’ among the many techniques.

However, each method does have some advantages and by using a combination of methods a better understanding and assessment of the flow of blood in the skin will be obtained. This in turn will lead to increased confidence in using measured values as a basis for patient treatment.

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172 I D Swain and L J Grant

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