Laser Doppler Imaging—A New Technique for Quantifying Microcirculatory Flow in Patients with Primary Raynaud's Phenomenon and Systemic Sclerosis

Microvascular Research 57, 284–291 (1999)Article ID mvre.1998.2124, available online at http://www.idealibrary.com on

Laser Doppler Imaging—A New Technique forQuantifying Microcirculatory Flow in Patientswith Primary Raynaud’s Phenomenonand Systemic Sclerosis

Stuart Clark, Fiona Campbell,* Tonia Moore, Malcolm I. V. Jayson,Terence A. King,† and Ariane L. HerrickRheumatic Diseases Centre, Hope Hospital, Salford M6 8HD, United Kingdom; †Department of Physics andAstronomy, University of Manchester, Manchester M13 9PL United Kingdom; and *Research andDevelopment Support Unit, Hope Hospital, Salford M6 8HD, United Kingdom

Received July 29, 1998

This was a pilot study to investigate the new techniqueof laser Doppler imaging (scanning laser Doppler) as atool to quantify microvascular blood flow in the digitsof patients with primary Raynaud’s phenomenon (PRP)and systemic sclerosis (SSc), and to determine in thefirst instance whether the flux patterns obtained differbetween patients with SSc, patients with PRP, andhealthy control subjects. Laser Doppler images of thedorsum of the hand and fingers were acquired at 23 and30°C in 17 healthy control subjects, 7 patients withPRP, 9 patients with the diffuse cutaneous variant ofSSc, and 24 patients with the limited cutaneous variantof SSc. Different flux parameters were compared be-tween groups. Analysis of variance found significantdifferences between groups in two tests: maximum dif-ference in flux between fingertips of the same hand at23°C (P 5 0.001) and maximum gradient in flux alonga finger (“distal– dorsal” flux difference) at 30°C (P 5

0.019). Post hoc tests highlighted the differences be-tween controls and patients with limited cutaneousSSc. This pilot study suggests that laser Doppler imag-
ing may allow objective measurement of microvascularflow in patients with PRP and SSc. This new technique
284

may overcome many of the problems inherent in sin-gle-channel laser Doppler equipment. © 1999 Academic Press

Key Words: systemic sclerosis; Raynaud’s phenome-non; laser Doppler imaging.

INTRODUCTION

Quantifying microcirculatory flow, in particular dig-ital microcirculatory flow, is one of the major chal-lenges confronting clinicians caring for patients withsystemic sclerosis (SSc). If we had a sensitive andreproducible technique of quantifying microcircula-tory flow, then we could measure digital microvascu-lar disease progression and/or responsiveness totreatment. This would facilitate clinical trials of treat-ment aimed at improving the peripheral circulation,which have so far relied on outcome measures whichare either subjective or insensitive to change.

None of the currently available techniques for mea-suring digital blood flow is ideal (Herrick and Clark,
1998). However, the relatively new technique of laserDoppler imaging (otherwise termed scanning laser
0026-2862/99 $30.00Copyright © 1999 by Academic Press

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Doppler), which is now being used in other areas ofmedicine to quantify microvascular flow, has the po-tential of being an exciting new tool in the assessmentof the digital microvasculature. Although laser Dopp-ler blood flow monitoring using a single probe hasbeen used in many studies of patients with primaryRaynaud’s phenomenon (PRP) and SSc (Herrick andClark, 1998), experience with the scanning laser Dopp-ler technique is very limited (Seifalian et al., 1994;Aghassi et al., 1995). Its advantages over single-chan-

el equipment are as follows:

1. Using a single delivery optical fibre and a singleollection optical fibre (as in most conventional laseroppler flowmetry systems), signals are collected

rom a hemisphere of approximately 1-mm radius.he laser Doppler flux measurements from these sys-

ems are prone to large variations due to the inhomo-eneous nature of the microcirculation coupled withhe small tissue volume probed by this technique. This

eans that there is considerable site-to-site variationn flux: even a small change in position or orientationf the probe can cause significant changes in estima-ion of blood flow. This means that two signals fromifferent sites (or signals from what is believed to be

he same site at different points in time) cannot beirectly compared. This limits the usefulness of the

echnique for monitoring change within an individualver time. In contrast, laser Doppler imaging buildsp a flux map by scanning the probe laser. These mapshow the inherent inhomogeneity of the microcircula-ion but allow averaging over regions of interest suchs the dorsum of the hand or the fingertips.2. Single point measurements are mostly obtained

sing contact probes which may in themselves alterlood flow. Laser Doppler imagers are noncontact ando avoid this problem.

Our aim was to perform a pilot study of laser Dopp-er imaging of the dorsum of the hand and fingers inatients with SSc at 23 and at 30°C, to determine in therst instance whether the flux patterns obtained dif-

ered from those of patients with PRP and healthyontrol subjects. Patients with PRP are generally con-
idered not to have underlying structural microvascu-ar disease (their problem is primarily vasospasm,
hich does not progress to irreversible ischaemia)lthough recently we have reported that subtle micro-ascular changes may occur (Bukhari et al., 1996).

MATERIALS AND METHODS

Patients

Patients and control groups were as follows:

1. Seventeen healthy control subjects (4 male, 13female; median age 37, range 23 to 61 years).

2. Seven patients with PRP (4 male, 3 female; me-dian age 41 years, range 21 to 67 years).

3. Nine patients with diffuse SSc (2 male, 7 female;median age 48 years, range 34 to 71 years).

4. Twenty-four patients with limited cutaneous SSc(3 male, 21 female; median age 45 years, range 30 to 64years).

Patients with PRP and SSc were all attending theRheumatology Department at Hope Hospital, Salford,and were randomly selected on the basis that theywere prepared to participate. All patients with PRPhad symptoms for at least 2 years, and none hadclinical or serological evidence of underlying connec-tive tissue disease. Of the 33 patients with SSc, 27fulfilled the criteria of the American Rheumatism As-sociation (Subcommittee for Scleroderma Criteria ofthe American Rheumatism Association Diagnosticand Therapeutic Criteria Committee, 1980). All 6 pa-tients who did not fulfill these criteria had Raynaud’sphenomenon: 4 in association with sclerodactyly (2were anticentromere antibody positive), 1 in associa-tion with digital pitting (and a positive anticentromereantibody), and 1 in association with digital ulceration,abnormal nailfold microscopy typical of SSc (and apositive test for antinuclear antibodies). All patientswith SSc suffered from Raynaud’s phenomenon. Pa-tients were divided into those with diffuse SSc andlimited cutaneous SSc on the basis of the extent of theirskin involvement: in patients with limited cutaneous
SSc skin involvement was confined to distal to theelbows, knees, and neck. One of the controls, 1 of the
Copyright © 1999 by Academic PressAll rights of reproduction in any form reserved.

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286 Clark et al.

patients with PRP, 1 of the diffuse SSc patients, and 3of the limited cutaneous SSc patients were smokers.Four of the diffuse SSc patients and 16 of the limitedcutaneous SSc patients were on vasodilator therapy,although patients were asked not to take vasodilatortherapy on the day of study. Also, all patients andcontrol subjects abstained from smoking and caffeine-containing beverages for 4 h prior to being studied.

Methods

Initially each patient/control subject acclimatised to23°C in a climate-controlled room. This is our stan-dard basal room temperature for studies in patientswith Raynaud’s phenomenon—colder temperatureswould be uncomfortable for many of the patientgroup. Laser Doppler images were then taken of thedorsal aspect of both of their hands, which were rest-ing on a table in front of the patient/control. Thedorsal aspect of both hands was then reimaged after20 min of acclimatisation at a room temperature of30°C.

Laser Doppler images were acquired with aMoorLDI-VR (Moor Instruments, Axminster, Devon,UK) which uses a helium–neon laser operating at 633nm. The unit incorporated an optional 45° mirror toallow hands to be imaged on a horizontal surface. Thelaser is raster scanned over a user-definable area withfaster scan speeds available at the expense of spatialresolution. For single, left, and right full-hand images,a resolution of 256 3 256 pixels which took around 5min to acquire was used. For repeated, fingers-onlyscans, a resolution of 64 3 40 pixels, for which onescan took 19.5 s, was used. Patients wore suitablesafety goggles at all times during the experiment andwere asked to remove all rings from their fingers, ifpossible.

The following parameters (chosen from experiencewith thermography) were derived for each subject at23 and 30°C:

1. The maximum difference in flux between the fin-gertips of one hand (the hand chosen was that in
which the greater maximum flux difference betweenfingertips was observed).

2. The lowest fingertip flux (lowest of the index,middle, ring, or little finger of either hand).

3. The average flux of the dorsum of the hand withthe lowest fingertip flux.

4. The maximum “distal–dorsal difference” in flux.This was the flux difference obtained by subtractingthe average flux of the dorsum of the hand from theflux of the lowest fingertip. Therefore this distal–dor-sal difference can be regarded as the flux gradientalong a finger, and for purposes of statistical analysisthe gradient of the finger demonstrating the lowestfingertip flux was chosen.

Figure 1 shows typical images from a healthy con-trol subject and from a patient with SSc.

Statistical Methods

Analysis of covariance, with sex as a covariate, wasused to analyse differences between groups, adjustedfor age and sex. The four groups of subjects (control,PRP, diffuse SSc, and limited cutaneous SSc) were firstanalysed as a categorical factor variable and second asa trend across groups indicating the increasing sever-ity of disease, with the order of the groups beinghealthy control, PRP, limited cutaneous SSc, and dif-fuse SSc. Dunn–Sidak post hoc tests were used tohighlight the main differences between groups when asignificant overall result was obtained.

RESULTS

Results are summarised in Table 1. They are ad-justed for age and sex. Analysis of variance foundsignificant differences between groups for two param-eters: maximum difference in flux between fingertipsof the same hand at 23°C (P 5 0.001) and maximum

istal—dorsal difference in flux at 30°C (P 5 0.019).Post hoc tests highlighted the differences betweencontrols and patients with limited cutaneous disease(Fig. 2).

The trend analysis showed a significant trend across
groups for four tests, a positive trend in maximumdifference in flux between fingertips of same hand at

FIG

.1.

Typ

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flux

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288 Clark et al.

23 (P , 0.0001) and 30°C (P 5 0.019), and a negativerend in the distal dorsal difference in flux at 23 (P 5.029) and 30°C (P 5 0.002).However, measurements at a single site did not

ifferentiate between groups. Although Table 1 showsseemingly statistically significant difference betweenroups for the dorsum of the hand with the lowestngertip flux at 30°C (P 5 0.017) post hoc tests pro-uced no differences between groups which were sig-ificant. This anomaly was due to an outlying pointhich “artificially” raised the mean of the PRP group.he P values for the trend in single-site measurementscross groups at 23 and 30°C were 0.276 and 0.073,espectively, for the lowest fingertip flux and 0.122nd 0.079, respectively, for the dorsum of the handith the lowest fingertip flux.

DISCUSSION

TABLE 1

Laser Doppler Fluxes in Healthy Control Subjects and Patients wit

Maximum difference influx between fingertips

of same hand Lowest finge

23°C 30°C 23°C

Controls(n 5 17)

39.1(25.5, 52.8)

53.5(36.0, 71.1)

53.5(39.1, 67.9)

PrimaryRaynaud’s(n 5 7)

50.1(29.3, 70.9)

79.0(52.3, 105.8)

49.6(27.6, 71.6)

Diffuse SSc(n 5 9)

61.4(42.6, 80.1)

65.5(41.4, 89.6)

38.3(18.5, 58.1)

Limitedcutaneous SSc(n 5 24)

79.1(67.8, 90.3)

84.8(70.4, 99.3)

45.7(33.8, 57.6)

P value fordifferencebetweengroups

0.001* 0.058 0.545

P value for trendacross groups

,0.0001* 0.019* 0.276

Note. Results are means (95% confidence intervals).* Significant differences on post hoc tests between control and l

control, PRP, diffuse SSc, limited cutaneous SSc).

Laser Doppler imaging has now been applied to theassessment of the microvasculature in a wide variety


of diseases/conditions, including monitoring of breastskin blood flow (Seifalian et al., 1995), wound healing(Eichhorn et al., 1994), assessment of burn depth (Naiziet al., 1993), and, of interest to the rheumatologist,measurement of hyperaemia over interphalangealjoints in patients with rheumatoid arthritis (Ferrell etal., 1996). The technique is noninvasive and almostcertainly will allow laser Doppler methodology to bemore fully exploited in many areas of medicine. Anadvantage over thermography is that laser Dopplerimaging measures blood flow directly, rather thanindirectly via temperature.

Ours was a pilot study involving small numbers ofpatients conducted within a limited time frame, theprimary aim being to see whether we could identifyblood flow patterns in patients with SSc. We believethat this study has demonstrated the potential useful-ness of laser Doppler imaging in the assessment ofmicrocirculatory flow in the hands and fingers of pa-tients with SSc and suggests that further studies arewarranted.

Limited Cutaneous SSc, and Diffuse SSc

x

Flux of the dorsum ofhand with lowest

fingertip fluxDistal–dorsal difference

in flux

23°C 30°C 23°C 30°C

05.2)

38.0(32.1, 44.0)

43.5(36.6, 50.3)

15.5(4.0, 27.0)

28.6(16.8, 40.3)

94.9)

46.1(37.0, 55.2)

58.0(47.5, 68.5)

3.5(214.0, 21.0)

19.9(2.0, 37.8)

48.5)

36.0(27.9, 44.2)

41.9(32.5, 51.3)

2.2(213.6, 18.0)

8.5(27.6, 24.7)

41.2)

45.5(48.1, 50.4)

53.8(48.1, 59.4)

0.2(29.3, 9.7)

6.6(23.1, 16.3)

9 0.082 0.017 0.182 0.019*

3 0.122 0.079 0.029* 0.002*

cutaneous SSc groups or significant trends across groups (order:

h PRP,

rtip flu

30°C

72.(58.9, 8

77.(57.8, 9

50.(32.3, 6

60.(49.5, 7

0.05

0.07

We examined the fingers at two temperatures, incase some differences between groups might be ap-

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FIG. 2. (a) Largest differences in flux between fingertips of the same hand at 23°C and (b) distal–dorsal differences in flux at 30°C showingtatistically significant trends across groups: healthy control subjects, patients with PRP, patients with diffuse SSc (DSSc), and patients withimited cutaneous SSc (LCSSc).


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B

B

D

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290 Clark et al.

parent only at 30°C (most patients are maximally va-sodilated at this temperature and so any fall in bloodflow due to sympathetic overactivity should be mini-mised). Two parameters which involved imaging ofmore than one area of the hand showed significanttrends across patient groups, whereas single-site mea-surements did not, demonstrating the value of thelaser Doppler imaging technique over single-probemeasurements. These two measures which look atdifferences in flux across sites (the difference in fluxbetween the distal phalanx and the dorsum of thehand and the greatest difference in flux between fin-gers of the same hand) are physiologically relevant.Regarding the distal–dorsal difference, in healthy con-trol subjects, microvascular flow is higher in the tips ofthe fingers than in the dorsum of the hand (and thefingertips are warmer), whereas this pattern may bereversed in patients with SSc. Therefore the flux gra-dient along the finger (and its responsiveness to a riseor fall in temperature) may be a useful parameter ofdigital vascular disease. The presence of a “negative”distal–dorsal flux gradient at 30°C was observed onlyin patients with SSc (although many patients with SScdid not have this negative gradient), suggesting that anegative flux gradient at 30°C might be a specific (butnot sensitive) test in identifying patients with under-lying structural vascular disease. Regarding the differ-ence in flux between fingertips of the same hand, inpatients with SSc the degree of structural digital vas-cular disease is unlikely to be identical between fin-gers, resulting in greater differences in blood flowbetween fingers than in patients with PRP and healthycontrols, as was demonstrated in this study and hasbeen demonstrated also indirectly by thermography(Darton and Black, 1991).

Our study confirmed clinical experience that themost severe digital vascular changes tend to occur inpatients with the limited cutaneous subtype of SSc—there is an association between anticentromere anti-body and digital ischaemia (Wigley et al., 1992).

Our findings suggest that these two parameters ofmicrovascular flow as assessed by laser Doppler im-aging (flux gradient along the finger and flux differ-ence between fingers) warrant further study. The first
step will be to assess their reproducibility: althoughwe are optimistic that the laser Doppler imaging tech-
F


nique will have better reproducibility than single-sitemeasurements (Bartelink et al., 1993), this cannot bessumed. The next step will be to measure microvas-ular flow before and after vasodilator therapy (forxample, prostacyclin). If improvement can be dem-nstrated in response to effective vasodilator therapy,hen laser Doppler imaging might be the long-awaitedbjective measure by which to monitor the microvas-ulature in patients with SSc, to be used in specialistentres with an interest in vascular flow. Anotherotentially useful application might be in helping to

dentify which patients presenting with Raynaud’shenomenon have structural vascular disease such asccurs in SSc (our study was too small to make anyonclusion about this, and there was overlap in resultsetween groups), although the technique of nailfoldicroscopy is already very helpful in this respect.

inally, we need to compare results obtained withaser Doppler imaging to those from other methods of

easuring blood flow in patients with Raynaud’s. Inhe first instance we need to compare results to thosef thermography. Future studies could include corre-

ations with capillary structure as measured by nail-old microscopy and with measures of digital arterylood flow.

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artelink, M. L., Wollersheim, H., Jansen, R. W. M. M., Theeuwes,A., and Thein, T. (1993). Reproducibility of the finger cooling test.Microvasc. Res. 45, 65–73.

ukhari, M., Herrick, A. L., Moore, T., Manning, J., and Jayson,M. I. V. (1996). Increased nailfold capillary dimensions in primaryRaynaud’s phenomenon and systemic sclerosis. Br. J. Rheumatol.35, 1127–1131.

arton, K., and Black, C. M. (1991). Pyroelectric vidicon thermog-raphy and cold challenge quantify the severity of Raynaud’sphenomenon. Br. J. Rheumatol. 30, 190–195.

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errell, W. R., Sturrock, R. D., Mallik, A. K., Abbott, N. C., Lockhart,J. C., and Edmondson, W. D. (1996). Laser Doppler perfusion

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Naizi, Z. B., Essex, T. J., Papini, R., Scott, D., McLean, N. R., andBlack, M. J. (1993). New laser Doppler scanner, a valuable adjunctin burn depth assessment. Burns 19, 485–489.

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Laser Doppler Imaging—A New Technique for Quantifying Microcirculatory Flow in Patients with Primary Raynaud's Phenomenon and Systemic Sclerosis