Laser Doppler Flowmetry and Transcutaneous Oximetry in Chronic Skin Ulcers: A Comparative Evaluation

Introduction/Objective. Laser Doppler Flowmetry (LDF) and transcutaneous oximetry (TcpO₂) are established methods for investigating cutaneous perfusion. To date, no study previously performed has compared data obtained from these 2 methodologies in cases of chronic cutaneous ulcers. Materials and Methods. Laser Doppler Flowmetry and TcpO₂ were performed in 25 consecutive outpatients with chronic lower limb ulcers (group A, experimental; 9 women and 16 men; mean age 67 years [range, 52–81 years]) and 25 age- and sex-matched healthy control subjects (group B, control) enrolled for the study. Ulcer aetiologies included 12 peripheral arterial occlusive disease, 9 chronic venous insufficiencies, and 4 pressure ulcers. Data were analyzed with Shapiro-Wilk and Wilcoxon-Mann-Whitney tests. Results. A statistically significant difference (P < .05) was found between LDF values of the 2 groups. No statistically significant differences were found between the 2 groups regarding the TcpO₂ measurements. Conclusion. The data confirmed the soundness of LDF while investigating local perfusion in patients with chronic cutaneous ulcers. The same diagnostic accuracy was not obtained by means of TcpO₂.

Chronic cutaneous ulcers of the lower extremities are a widespread cause of morbidity and mortality worldwide.^1-2 The main etiologies are chronic venous insufficiency, peripheral arterial occlusive disease (PAOD), pressure ulcers (PU), and diabetes.^3-5 Chronic venous leg ulcers (VLUs) affect about 1% of the adult population in Europe and North America.⁴ Proper tissue oxygenation is essential for wound healing. Oxygen deficits were found in VLUs, PUs, PAOD, and diabetic foot ulcers (which are often associated with PAOD).⁶ Indeed, according to the literature, cutaneous microcirculation has gained increased interest in the pathogenesis of skin ulcers.^7-13

Laser Doppler Flowmetry (LDF) is an established method for measuring the microcirculatory blood flow using a fiber optic probe. This method is a continuous, noninvasive, real-time assessment of skin perfusion¹⁴; it measures the total local microvascular flow. The LDF method is based on the Doppler shift of emitted laser light travelling through tissue and reflecting off moving objects such as circulating red blood cells. The LDF signal is a stochastic representation of the number of erythrocytes in sample volume multiplied by their velocity, referred to as flux. Since the red blood cell flux is linearly correlated with skin blood flow, it is taken as an estimation of blood flow; for this reason, the flux signal obtained by LDF is usually expressed in arbitrary perfusion units. The proposed technique is potentially applicable to the detection of specific pathological aspects of microcirculation, such as arterial occlusion in the leg, diabetes mellitus, and congestive heart failure, where the venous-arteriolar reflex may be affected.

Transcutaneous oximetry (TcpO₂) is a standardized procedure used to measure oxygen pressure in a given tissue to determine its level of oxygenation. It is useful to assess skin microcirculation since the observed values can be predictive of wound healing. Other applications are the evaluation of the need for limb amputation and its level and the effect of revascularization procedures¹⁵; TcpO₂ is also finding increasing application as a diagnostic tool to assess the level of oxygenation of skin ulcers and cutaneous flaps. Measurement is performed by application of an electrode in the adjacent area of a wound and results are expressed in mm Hg.¹² While the TcpO₂ reference values are now standardized,^15,16 reference values for LDF in patients with chronic cutaneous ulcers have not yet been identified.^17,18 

The aim of this study was to investigate and compare baseline values of LDF and TcPO₂ in patients with chronic lower extremity skin ulcers and test the soundness of these methodologies in wound healing assessment studies.

Twenty-five consecutive outpatients (group A, experimental) with chronic lower limb ulcers (9 women and 16 men; mean age 67 years [range, 52–81 years]) followed up at the Cutaneous, Mini-invasive, Regenerative, and Plastic Surgery Unit, Parma University Hospital (Parma, Italy), and 25 healthy control subjects, age and sex matched (group B, control), were enrolled for the study. Ulcer etiologies were 12 PAOD, 9 chronic venous insufficiencies, and 4 PUs. The trial was conducted in compliance with the Declaration of Helsinki and the Guidelines for Good Clinical Practice; all enrolled patients provided written informed consent before inclusion in the study. The analyses were carried out by a trained operator after 30 minutes of acclimatization at a temperature of 24 ± 1°C, as suggested by Ambrózy.¹⁷ Measurements were performed (in group A) after a 24-hour or more interval following ulcer debridement and adjacent skin, thus avoiding the influence of local skin trauma on examination. 

Digital blood flow was measured by a laser Doppler flowmeter (PeriFlux System 5000, PF 5001–5010 units, standard probes 407–D826; Perimed AB, Järfälla, Sweden); the laser Doppler apparatus was connected via RS232 interface with a PC Notebook (Hp Compaq CQ58; Palo Alto, CA) with online analysis of information on skin blood flow by PeriSoft (PSW-ExM; Perimed AB) software. The probe was positioned at the most distal point of the ulcer margin (Figure 1). The overall test duration was 25 minutes; measurements were performed for 20 minutes in supine position and then with legs in declivous position for 5 minutes. 

In the same sessions, transcutaneous oxygen tension was measured by placing the laser Doppler flowmeter probe close to the most proximal point of the ulcer margin. The measuring site was cleaned carefully using saline solution, and the transducer was fixed to the skin with double-sided adhesive rings and contact liquid supplied by the manufacturer. Calibration period was 10 minutes, and the TcpO₂ signal was continuously recorded for 20 minutes. 

Although perfusion may vary between such points, the investigators chose to place the 2 probes (LDF distal and TcpO₂ proximal) in order to try to standardize the procedure. Each time, volunteers from group B had probes placed on the same sites as patient equivalents from group A (matched by sex and age). They then compared the values observed in the 2 groups to determine whether there were differences in LDF and TcpO₂ reference values. 

As shown in Table 1, the investigators found for group A that mean LDF values were 31 perfusion units, and the mean TcpO₂ values were 28.44 mm Hg. For group B they found that mean LDF values were 14.12 perfusion units, and mean TcpO₂ values were 28.56 mm Hg. 

Table 2 illustrates the Shapiro-Wilk test results, whereby it is possible to check whether a sample comes from a normally distributed population. The Shapiro-Wilk test was chosen because it has the best power for a given significance. This test is based on the correlation between data and normal scores, which are order expectations of a normally distributed sample: a low correlation between these 2 quantities indicates non-normality. If the P value is lower than the chosen alpha level (.05 in this case), the null hypothesis of normality is rejected, and there is evidence that the data are not from a normally distributed population. All variables considered showed provenance from a non-normal population. 

The Wilcoxon-Mann-Whitney test results are shown in Table 3. The P values reported were calculated with the Monte Carlo procedure, justifiable in this case because despite asymptotic distribution, small sample sizes made the approximation unreliable.¹⁹ The empirical P value was calculated as follows:

Where M equals the number of samples with (Test Statistic ≥ t), N equals the total number of samples, and T equals the observed Test Statistic.

Procedures that use M/N generally tend to slightly underestimate the P value if the null hypothesis is true. To avoid the anticonservative nature of this measure, a value of 10 000 was used for the total number of samples. The formulas used to calculate the empirical P value and its confidence interval are given in the appendix. A significant difference between group A and group B was detected for “Laser Doppler” variables.

Box plots of the considered variables divided between the experimental and control groups are reported in Figure 2 (a “case” variable value which equals 0 refers to the control distribution; a value equal to 1 refers to case distribution). The graph highlights the same conclusions obtained from the Wilcoxon test: group A and group B had different distributions compared for LDF. No statistically significant differences were found between the 2 groups when considering TcpO₂ measurements. 

Chronic ulceration of the lower limb is a frequent condition with a prevalence of 3% to 5% in individuals aged more than 65 years.²⁰ Many factors contribute to the pathogenesis of leg ulcers. The main causes are chronic venous insufficiency, PAOD, PUs, and diabetes.³ 

Reliable information as to proper diagnosis of nonhealing wounds requires the assessment of both macrocirculation and microcirculation. This is particularly important in the view of the promising clinical use of mesenchymal stem cells in regenerative medicine as an aid to enhance local perfusion.^21-30 Macrocirculation can be assessed using ultrasound, angiography, and peripheral pressure indexes. Microcirculation can be evaluated by means of LDF and TcpO₂. Laser Doppler Flowmetry was first introduced by Stern³¹ in 1975, thereby permitting a continuous, noninvasive study of microcirculation. Measuring depth is about 0.5 mm to 1 mm, reaching the superficial vessels: arterioles, venules, shunts, and capillaries. In accordance with the “European Laser Doppler Users Groups,” the LDF output is semiquantitative and expressed in perfusion units of output voltage (1 perfusion unit = 10 mV).³² Laser Doppler Flowmetry has both great diagnostic and prognostic potential with regard to its possible involvement in monitoring therapies that aim to promote wound healing by stimulating the processes of local angiogenesis; however, because of the complex structure and random orientation of the cutaneous microcirculation, the obtained measurements are only semiquantitative and relative. 

The limitations of the present study consist of the small sample size and the inclusion of ulcers of various etiologies. Nevertheless, according to the investigators’ data, a specific range of reference values have been identified in patients with chronic skin ulcers, a range that is statistically different from the normal population. Conversely, no statistically significant differences have been found between TcpO₂ values in patients with lower extremity ulcers and controls. 

Microcirculation is a dynamic system with numerous variations within the normal value range. Laser Doppler Flowmetry is an excellent noninvasive technique for the measurement of skin blood flow; an advantage of LDF over other methods is that it gives a good opportunity for direct, real-time assessment of microvascular function. The results presented herein confirmed the soundness of this approach, showing significant differences in local flow data between patients with or without ulcers. The same accuracy was not obtained by means of TcpO₂.  

P^{^}mc = estimated Monte Carlo P value.

The variable M has a binomial distribution with N trials and success probability P. The asymptotic standard error is calculated as follows:

The confidence interval is the following:

Where zα/2 is the 100(1-α/2)^th percentile of standard normal distribution.

Affiliations: Department of Medicine and Surgery, Plastic Surgeon Section, University of Parma, Parma, Italy; and the Cutaneous, Mininvasive, Regenerative and Plastic Surgery Unit, Parma University Hospital, Parma, Italy

Correspondence:
Edoardo Raposio, MD, PhD
Director, Plastic Surgery Chair and Residency Program
Department of Medicine and Surgery
University of Parma
Via Gramsci 14
43126, Parma, Italy
edoardo.raposio@unipr.it

Disclosure: The authors disclose no financial or other conflicts of interest.