Transcutaneous oxygen measurement using ratiometric fluorescence imaging as a valid method for monitoring free flap transplants

Abstract

BACKGROUND:

Reconstruction of soft tissue defects with free flaps is a common procedure in plastic and reconstructive surgery. Most postoperative complications occur within the first 48–72 hours after surgery. After postoperative complications, short perfusion restoration times may improve flap survival rates by up to 30–50%. Ratiometric fluorescence imaging is an additional or alternative method of postoperative flap monitoring.

OBJECTIVE:

To test the efficacy and utility of transepidermal oxygen flux imaging to evaluate postoperative skin oxygenation of free and local flaps in the first 48 hours after surgery.

METHODS:

The study included 32 patients (aged between 18 and 80 years; mean age 52.9) with a tissue defect covered with a free flap transplant at the Department of Plastic and Reconstructive Surgery of the University Medical Center Regensburg. Postoperative oxygen flux was measured with the ‘VisiSens system’ placed on the vascular pedicle as well as on the peripheral and central part of the flap.

RESULTS:

Values of oxygen flux were higher in case of flap congestion (0.069±0.012) or flap necrosis (0.155±0.083) than in cases without any complications (0.061±0.006). Flux values of different areas of the same flap showed only minimal differences (central part: 0.065±0.008, peripheral part: 0.070±0.009, vascular pedicle: 0.056±0.004); the level of significance was p = 0.904.

CONCLUSION:

Imaging transepidermal oxygen flux by ratiometric luminescence seems to be a reliable alternative, indirect method of postoperative flap monitoring with regard to microcirculatory function and flap viability.

Keywords

Postoperative free flap monitoring microcirculatory function tissue oxygenation tissue perfusion flux imaging flap monitoring transcutaneous oxygen measurement

1 Introduction

Free flap transplantation of large soft tissue defects after burns, serious trauma, tumor resection or congenital anomalies is a common procedure in plastic and reconstructive surgery. Even with optimal planning and preparation, the complication rate after free flap transplantation may be as high as 20% [1 –3]. The decisive factor for the success of an autologous tissue transplantation is the integrity of vesicular anastomosis. If the time from the occurrence of the complication until restoration of sufficient perfusion is kept as short as possible, adequate revision procedures may improve flap survival rates by up to 30–50% [4]. Complications predominantly develop within the first 72 hours after surgery, hence more intensive monitoring is required during this period [5]. The highest risk to the vitality of transplants is impaired venous perfusion, which may be caused by the complete occlusion of venous anastomosis or by microthrombosis in the capillary structures. In contrast, flap transplants are less frequently affected by reduced arterial perfusion. Assessing the microcirculatory function of flaps by means of objective monitoring methods may help identify potential complications at an early stage, even prior to the development of clinical signs of reduced flap perfusion such as changes in capillary refill, color, temperature or turgor.

Direct evaluation of microcirculatory function by means of imaging techniques such as magnetic resonance imaging (MRI) or positron emission tomography (PET) is rather complex, so that both methods have not yet been established as clinically routine in this context. Measuring methods such as infrared or white light spectroscopy allow the indirect evaluation of tissue perfusion by determining the extent of oxygenated hemoglobin in the tissue. However, only small measurement areas can be evaluated, and the use of individual intensity measurements facilitates local measurement fluctuations and errors.

Transcutaneous oxygen measurement using fluorescent optical sensors has not only yielded very reliable measurement results in several clinical studies but has also been shown to be less subject to local measurement fluctuations [6 –9]. Based on these first examinations, further studies have focused on the use of fluorescent optical sensors to expand their use in daily clinical practice [10, 11]. These studies have shown that flap perfusion may be reliably evaluated across larger measurement areas, thus allowing the early identification of any complications.

The objective of this study was to investigate whether transcutaneous oxygen measurement using flux imaging presents a reliable technique for postoperative monitoring of free flap transplants and for the early detection of possible perfusion disorders in the flap vessels.

2 Material and methods

2.1 Study design

The study cohort consisted of 32 patients aged between 18 and 80 years who had received a free flap transplant at the Department of Plastic and Reconstructive Surgery of the University Medical Center Regensburg between 2013 and 2015. The exact composition of the patient cohort is shown in Table 1.

Table 1
General data of the included patients

Gender of the patients Men n = 19

Women n = 13

Mean age of the patients 52.9 years (SD±18.4)

Cause of the tissue defect Trauma 31.3%

Infection 28.1%

Carcinoma 21.9%

Decubitus 9.4%

Body area of the tissue defect Lower extremity 40.6%

Buttocks 21.9%

Chest or abdominal wall 15.6%

Upper extremity 12.5%

Back 6.2%

Face 3.1%

Complication rate No complications 63.3%

Minor complications 30.0%

Major complications 6.7%

Gender of the patients	Men	n = 19
Mean age of the patients	52.9 years (SD±18.4)
Cause of the tissue defect	Trauma	31.3%
	Infection	28.1%
	Carcinoma	21.9%
	Decubitus	9.4%
Body area of the tissue defect	Lower extremity	40.6%
	Buttocks	21.9%
	Chest or abdominal wall	15.6%
	Upper extremity	12.5%
	Back	6.2%
	Face	3.1%
Complication rate	No complications	63.3%
	Minor complications	30.0%
	Major complications	6.7%

2.2 Measuring method

Over the first 48 postoperative hours, transcutaneous oxygen was measured in the area of the vascular pedicle as well as in the peripheral and central part of the flap by means of fluorescent optical sensor foils (PreSens, Regensburg, Germany) (Fig. 1). The sensor foils were based on the principle of dynamic fluorescence quenching. A central part of sensor foils is a polymer indicator solution that is applied onto a carrier foil. This foil also acts as a barrier to the surrounding oxygen of the atmosphere so that changes in the emitted fluorescent radiation may only be caused by the exchange of oxygen between the skin and the indicator medium. The sterile sensor foil is directly applied to the skin, and the indicator molecules are induced via a light-emitting diode (LED) light source. The emitted fluorescent radiation is detected with an integrated camera. High oxygen concentrations in the tissue lead to increased oxygen flux into the indicator medium and thus to the reversible reduction of fluorescent radiation.

Fig.1

Fluorescent optical sensor foil on a heel, physician holding sensor.

An indicator (fluorophore) is excited to a high excitation stage via a light source. In the presence of a quencher, for instance oxygen, the excited energy of the indicator becomes non-radiating and is reversibly transferred to the oxygen of this quencher. The remaining fluorescent radiation is detected with a camera and allows conclusions about the present concentration of the quencher oxygen [12, 13]. In comparison to simple intensity measurements [14], methods of measuring the duration of fluorescent radiation (lifetime imaging) [15] or fluorescent radiation in proportion to a reference dye (ratiometric imaging) [16 –18] yield more stable results and are less susceptible to inhomogeneous distributions of indicator molecules on the sensor foil. Sterile planar sensor foils (VISI Sens measuring system, PreSens, Regensburg, Germany) that are directly applied onto the transplant are a key component of the measurement tool.

In ratiometric luminescence imaging (RLI), for instance, a red and a green fluorescent dye, as oxygen-sensible indicator and reference dye respectively, are dissolved in a polymer matrix. The polymer sensor matrix is subsequently applied onto an oxygen-impermeable polyester, thus generating the sensor foil for RLI measurement. The exact composition of the individual components of the polymer sensor matrix (porphyrin (indicator dye) and aminonaphthalimide (reference dye), etc.), guarantees exactly the same measurement results in different sensor batches, thus making the calibration of the system unnecessary. A hand-held device that is connected to a mobile computer via a USB port contains both an LED light source for exciting the indicator dye as well as a camera for detecting the corresponding fluorescent radiation. Depending on the field of application, camera heads with measurement units of different sizes may be used (for instance, to monitor perfusion in the case of finger amputation) (Fig. 1). The two fluorophores are excited by means of the LED light source; their fluorescent radiation is evaluated via a Red-Green-Blue (RGB)-chip and set in relation to each other. In oxygen flux imaging, a calculation of the ratio between the indicator dye and the reference dye at different measurement times generates a gradient. Low oxygen concentration in the tissue results in an increased oxygen gradient between the sensor foil and the skin and thus in increasing oxygen diffusion from the foil into the skin. Decreased oxygen concentration in the foil and the resulting decrease in the quencher oxygen lead to higher fluorescent radiation of the indicator. At the same time, the radiation intensity of the reference dye remains unchanged, increasing the ratio between the indicator dye and the reference dye. Depicting the ratio between the two fluorophores in relation to successive individual measurements yields a monotonically increasing graph with steep gradient. In contrast, high oxygen concentration in the tissue only leads to low oxygen diffusion from the sensor foil into the tissue. The graphical representation shows a relatively low increase in the ratio between the indicator dye and the reference dye and thus a flatter curve.

Besides the absolute values, the two-dimensional color-coded presentation of oxygen flux facilitates the identification of insufficiently perfused tissue areas. Insufficient tissue perfusion and the associated increase in oxygen flux from the sensor foil into the skin increasingly changes the fluorescent radiation of the indicator, thus leading to color changes in 2D-mapping; in contrast, increased tissue perfusion and thus lower oxygen flux only causes discrete changes in color-coded mapping.

All patients were informed about the study content and gave their written consent. The study was approved by the local Ethics Committee and conducted according to the guidelines stated in the Declaration of Helsinki.

2.3 Statistical analysis

Data were analyzed by means of a t-test to evaluate whether the level of oxygen flux was significantly associated with the development of complications. An analysis of variance (ANOVA) was conducted to find any differences in oxygen flux between the groups ‘no complications’, ‘flap congestion’, ‘flap necrosis’, and ‘infection’. The different types of complications were compared pairwise with regard to differences in oxygen flux by means of post-hoc tests. Boxplots as well as bar charts depicting mean values and 95% confidence intervals as error bars were used for graphical presentation.

A t-test was conducted to check for differences in oxygen flux between the peripheral and the central part of the transplant. Differences in oxygen flux between the pedicle and the central and peripheral part of the flap were investigated using an ANOVA. The oxygen flux of the different body sites was compared by means of post-hoc tests. Boxplots as well as bar charts depicting mean values and 95% confidence intervals as error bars were used for graphical presentation.

Differences in the development of complications and the demographic factor, sex and age, were analyzed by means of Fisher’s exact test and presented by a contingency table. Any relationships between complications and patient age were analyzed with a t-test.

3 Results

3.1 General results

Mean age of the study group (n = 32, 19 men and 13 women) was 52.9±18.4 years.

Indications for flap transplant surgery were as follows: 10 (31.3%) patients had suffered a trauma including a soft tissue defect or subsequent impaired wound healing, 9 (28.1%) patients had developed infection, 7 (21.9%) patients had undergone surgery because of carcinoma, and 3 (9.4%) patients had required flap transplants because of extensive decubitus. The indications of the 3 remaining patients (9.4%) did not fit into any of the above-mentioned categories.

The majority of soft tissue defects were found on the lower extremity (13 patients, 40.6%) followed by the buttocks, the sacrum, and the small pelvis (7 patients, 21.9%). Five patients (15.6%) had required transplants on the chest or the abdominal wall, 4 patients (12.5%) on the upper extremity, 2 patients (6.2%) on the back, and 1 patient (3.1%) on the face. Two patients were excluded from analysis because of unusable data due to the development of dry skin necrosis that resulted in poor adhesion of the sensor foil. For this reason, the statistical analysis included only 30 patients.

Nineteen patients (63.3%) did not develop any postoperative complication. Six patients (20.0%) showed venous congestion that was successfully treated with local therapeutic measures such as the application of leeches. Three patients (10.0%) with local infection either received antibiotic treatment or surgical debridement to avoid loss of the transplant. Complete flap loss was observed in 2 patients (6.7%). Overall, 9 patients (30.0%) showed minor complications and 2 patients (6.7%) major complications, whereas the remaining 19 patients (63.3%) did not develop any complication (Table 1).

3.2 Transcutaneous oxygen measurement as a method of postoperative flap monitoring

3.2.1 Comparison of oxygen flux according to body site

Mean flux values were analyzed by means of ANOVA in dependence of the respective measurement area. Values to be analyzed were divided into central and peripheral measurements as well as measurement on the pedicle. Flux values of different areas of the same flap showed only minimal differences (central part: 0.065±0.008, peripheral part: 0.070±0.009, vascular pedicle: 0.056±0.004); the level of significance was p = 0.904. (Fig. 2)

Fig.2

Comparison of oxygen flux according to body site.

3.2.2 Comparison of oxygen flux according to complication

As expected, the comparison of mean flux values between uneventful clinical course (0.061±0.006) (Figs. 2 and 3) and the development of postoperative complications (0.079±0.016) showed an increase in oxygen flux and insufficient microcirculatory function (Figs. 3 and 5); however, the results did not show any statistical significance in the t-test (p = 0.196). A significant result (p = 0.042) was found in the comparison of the different groups (no complication, congestion, necrosis, and infection) by means of univariate ANOVA. Values of oxygen flux were higher in the case of flap congestion (0.069±0.012) or flap necrosis (0.155±0.083) than in cases without any complications (0.061±0.006). Similar high values (0.061±0.030) were found for infected transplants and uneventful clinical courses (0.061±0.006). (Figs. 3–6)

Fig.3

Average of ROI of a well perfused flap (Fig. 4).

Fig.4

Well perfused free flap transplant.

Fig.5

Average of ROI of a free flap with venous congestion (Fig. 6).

Fig.6

Free flap transplant with venous congestion.

3.2.3 Comparison of complications according to sex and age

The occurrence of complications was not associated with the demographic factor sex of the patients (p = 0.621). The t-test comparing complications with regard to age had a level of significance of p = 0.596; this result does not indicate any association between age and the development of complications.

4 Discussion

The progress and success of microsurgery over the past few decades has increased the number of free flap transplants in various fields of medicine. Despite improved surgical techniques and technical innovations, the success rate of free flap transplants has remained stable at a level of about 90–95% for many years [19 –21]. Early revision surgery may guarantee surgical success and prevent loss of transplant in up to 50% of complications [4].

In our study, the 30 free flap transplants investigated showed minor complications in 9 cases and major complications (total loss of transplant) in 2 cases. Six complications (20.0%) were venous congestion, 3 (10.0%) infection, and 2 (6.7%) loss of transplant due to necrosis, whereas 19 transplants (63.3%) showed an uneventful clinical course. Thus, our complication rate of 36.7% was similar to the rate found by Giunta et al., who described a complication rate of 40.0%, a transplant success rate of 86.0%, complete loss of transplant rate of 6.0%, and a partial loss of transplant rate of 9.0% [22].

Clinical evaluation of the transplant according to capillary refill, color, temperatures or turgor is still the gold standard for assessing the vitality of flaps [23 –35]. Although direct measurement methods such as PET or MRI allow the exact evaluation of flap perfusion, the relatively high amount of time and effort required is not justified in every postoperative phase. Further problems may be patient transport or artefacts due to implants.

Ultrasound examination with a contrast medium is a further direct measurement method. Because of its very good spatial resolution, this method is very suitable for evaluating flap perfusion [26 –29]; however, contrast medium has to be applied for each single measurement, and evaluating the examination requires some experience.

In contrast to direct measurement methods of depicting microcirculatory function, indirect measurement methods are based on the evaluation of metabolites such as oxygen or oxygenated hemoglobin. Measuring oxygen flux is an indirect measurement method; its advantage over direct measurement methods is the simple measuring procedure that only lasts about 20 seconds and does not require any previous calibration. Additionally, this method is marked by unrestricted mobility.

One of the most common methods is laser Doppler flowmetry; however, the huge disadvantage of this method is its high susceptibility to motion artefacts and the complex calibration of the laser prior to measurement [30 –32].

Other less susceptible indirect measurement methods such as infra-red or white light spectroscopy have only low local resolution, allow the evaluation of only small measurement areas, or are susceptible to large local measurement fluctuations or measurement errors because of the requirement of individual intensity measurements.

In contrast to the indirect measurement methods described above, oxygen flux measurement allows the fast evaluation of tissue perfusion without any previous calibration and is not susceptible to motion artefacts. A further method of indirect evaluation of microcirculatory function is the Clark electrode [33], which requires calibration of the sensor, as in the case of laser Doppler flowmetry.

Measurements with fluorescent optical sensors do neither require calibration nor oxygen. Additional systematic errors such as light reflections or inhomogeneous distribution of the indicator molecules may be eliminated by means of ‘lifetime imaging’ or ‘ratiometric imaging’. This method has already been shown to yield very good results in evaluating flap vitality in several clinical studies [7 , 35]. Additionally, the high local resolution and the 2-dimensional color-coded presentation of oxygen flux allow the exact evaluation of border areas such as flap borders. This way, insufficiently perfused areas may not only be easily perceived visually, but the oxygen flux of the respective areas may be calculated separately; thus, false positive results in border areas are significantly reduced. Further advantages of flux imaging are its ease of use and the arbitrary size settings of the sensor foil. Furthermore, sensor foils are a cost-efficient, sterile, disposable item that may be separately shrink-wrapped by means of gamma sterilization.

Oxygen flux monitoring also guides the early identification of minor complications that could be treated by appropriate minor surgical intervention such as evacuation of hematoma or vacuum-assisted closure-therapy or by conservative treatment such as elevation of the affected limb, heat, or the application of leeches. There is sufficient evidence that the early identification of insufficient microcirculatory function significantly contributes to flap survival [19, 36]. The highest difference in values compared to the control group was found in the group ‘major complications’ such as necrosis that resulted in loss of transplant (6.7%). However, this group (n = 2) was not large enough for significant statistical analysis.

Oxygen flux measurement did not yield any significant differences with regard to the affected body site. Yet, oxygen flux was lower in the first 48 hours after surgery because of reactive hyperemia. Early postoperative hyperemia has also been observed when using other methods, for instance laser Doppler flowmetry or micro-light guide spectrophotometry [37, 38]. However, early postoperative hyperemia may conceal early flap infection that is also marked by hyperemia. Thus, clinical evaluation of the flap remains essential, also during the early phase.

Oxygen flux monitoring did not yield any significant differences between men and women, age groups, or types of flaps; thus, this method may be viewed as a generally applicable and immediately evaluable indirect tool in the multitude of reconstructive surgical interventions in the field of plastic surgery.

5 Conclusion

In summary, flux imaging represents a good alternative to established monitoring systems in the context of flap transplantation surgery. This measuring system allows the early identification of insufficient microcirculatory function and the coverage of relatively large tissue areas. These advantages as well as the system’s easy learnability and use allow fast and uncomplicated postoperative monitoring. Furthermore, flux imaging represents a non-invasive method. However, this method should be further developed to eliminate external confounding factors and to be able to examine larger patient cohorts. This way, sufficient data may be collected to specify universal threshold values to indicate the point in time when a transplant is at risk because of insufficient microcirculatory function. In contrast to established methods of postoperative flap monitoring, flux imaging is easily performed in addition to clinical examination.

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Gender of the patients	Men	n = 19
	Women	n = 13
Mean age of the patients	52.9 years (SD±18.4)
Cause of the tissue defect	Trauma	31.3%
	Infection	28.1%
	Carcinoma	21.9%
	Decubitus	9.4%
Body area of the tissue defect	Lower extremity	40.6%
	Buttocks	21.9%
	Chest or abdominal wall	15.6%
	Upper extremity	12.5%
	Back	6.2%
	Face	3.1%
Complication rate	No complications	63.3%
	Minor complications	30.0%
	Major complications	6.7%