Applying spectrochemical analyses on venous disease patients treated by N-Butyl Cyanoacrylate Ablation Surgery

2.1 Ethics

This study was approved by the Ethics Committee of the Medical school of Istanbul University-Cerrahpasa, Cerahpasa, Istanbul, Turkey (permission number: 1787, date: 12.04.2019). Two expert physicians working in the field of clinical Department cardiovascular surgery in the clinic of Istanbul Training and Research Hospital were provided the information and biological materials of the chronic venous insufficiency diagnosed patients. A total of 18 blood serum samples were required in this study before and after CAE surgery. Peripheral blood samples were collected from both two groups. The data collected from patients who were involved in this experiment were subject to the Personal Data Act and Act on Ethical Review for research involving human subjects. All participants were fully informed about the study and signed consent forms prior to participation.

2.2 Study design

From January 2019 to January 2020, 18 patients who underwent the CAE technique of the great saphenous vein (GSV) under local anesthesia due to chronic venous insufficiency were included in this study. Ablation treatment with endovenous cyanoacrylate was applied to patients with C2 to C4 grade venous insufficiency, according to the CEAP clinical classification. The treatment is contraindicated in patients who have a history of deep venous thrombosis, have femoral vein insufficiency, thrombophilia, known sensitivity to cyanoacrylate adhesives the presence of severe systemic disease, and in pregnant patients. This procedure was not used in any patient who had any of these conditions [21]. Inclusion criteria were as follows for patients: Age ⩾ 35 years and ⩽ 70 years and able to give informed consent for the procedure. CEAP class C2–C4 varicose veins. Symptomatic primary or recurrent GSV incompetence with reflux $>$ 0.5 sec. on color Duplex ultrasound including one or more of the following symptoms: aching, throbbing, heaviness, fatigue, pruritus, night cramps, restlessness, generalized pain or discomfort, and swelling. Patients who had GSV diameters of 5.5 mm to 12 mm in the standing position and contained a refluxing segment of at least 10 cm. Detailed demographic data of the patients who were treated using endovenous NBCA ablation therapy was collected. Whole blood count, RBC (red blood cell), WBC, PLT. Sedimentation rate, CRP (C-reactive protein), ALT (alanine aminotransferase), AST (aspartate aminotransferase), urea, creatinine was studied in all patients on admission to the clinic before the operation. These examinations were repeated after two weeks from the operation. Clinical diagnosis is represented in Table 3.

Under local anesthesia, in the operating room, an appropriate segment of the saphenous vein was selected under CDUS and a 5F introducer sheath was placed. The same embolizing agent system was used in all patients. Catheter placement was confirmed by ultrasonography. The catheter of the system was sent through approximately 3 cm distal to the saphenofemoral junction and a 0.035-inch guidewire was sent through the sheath. Ultrasonography was used to determine whether the guidewire reached the saphenofemoral junction and a 4F delivery catheter was inserted into it. The catheter was confirmed to be at the saphenofemoral junction and then draw back 3 mm and a 3 ml injector and plunger system provide the NBCA injection, was positioned. The catheter location was rechecked by ultrasonography and it was confirmed that it was not at the saphenofemoral junction. The connection point in the vein was compressed and obstructed with an ultrasonography probe. As the injector advanced through the saphenous vein, 0.3-ml NBC was dispensed into the vein at each beat, and compression was performed at the same time. The catheter was withdrawn in the vein at a speed of 2 cm/s and the procedure was continued. Compression was applied for 1 min at the end of the procedure. It was shown by ultrasonography that the saphenofemoral junction was open, and then the procedure was terminated after the rest of the great saphenous vein was occluded [22, 23]. Patients were discharged the same day.

2.4 Sample preparation for the acquisition of ATR-FTIR spectra

The FT/IR-4700 (JASCO, Tokyo, Japan) Mid-IR spectrometer equipped by attenuated total reflection (ATR) TM crystal plate, with liquid-nitrogen cooled mercury cadmium telluride (MCT) contains diamond with 32 running spaces at a resolution of 4 cm - 1 with zero-filling of the interferogram giving 4 cm - 1 data spacing corresponding to 3684 data points. the infrared spectrum was recorded in the 4000 cm - 1 to 600 cm - 1 regions. Three replicates from each sample were obtained and the average spectra were used. Firstly, the collected blood samples were centrifuged (15 min., 3000 rpm) then serum was separated and stored ( - 80 ∘ C) till spectroscopic measurements. Samples were dried as in the earlier studies [24, 25]. To avoid extensive water absorbance, the empty diamond/MCT of the ATR unit was recorded as background and subtracted repeatedly before each sample (1 μ L) was laid onto IR-reflective glass one by one for homogeneity [26]. The acquisitions were made three times per sample. The crystal of the device was washed separately for each sample’s measurements. Flowing the preprocess, all spectral recordings were analyzed via JASCO Spectra Manager version 2 and OPUS software. The features were baseline and ATR corrected, vector normalized, 25 points smoothed with Savitzky-Golay from 4000 to 600 cm - 1 bands and averaged [27]. We used Thermo Scientific’s GRAMS AI software to analyze the protein secondary structure through curve fitting. After smoothing two consecutive points, we calculated the second derivative spectrum from the ATR-FTIR spectrum. Judging from the peaks above 1750 cm - 1 , the absorption band at low wave numbers had no water vapor characteristics. We subtracted the straight baselines passing through the ordinates of 1700 cm - 1 and 1610 cm - 1 before curve fitting. We modified the baseline again via the least squares curve fitting software which allows to adjust, the horizontal baseline as an additional parameter to obtain the best fit. We used the second derivative spectrum, in order to determine the initial peak position of the curve fitting. Also, the Gaussian function is used to fit the peak. We considered the area under the entire band as 100%, and each component after fitting is expressed as a percentage fraction [28].

2.5 Statistical analyses for FT-IR spectra

The statistical significance of the calculated values absorbance was analyzed by one-way ANOVA followed by the Tukey’s test (Statistica 10). The obtained experimental results were represented as the means ± SEM (the standard error of the mean). Moreover, to obtain information about the correlation between obtained FTIR spectra and laboratory condition results, the Pearson’s test was performed with 0.05 and a significance level of 95%. All analyses were performed using Past 3.0 software. Firstly, group comparisons were performed concerning to determine CEA surgery before and after effects who suffered from varicose veins and treated by n-butyl cyanoacrylate ablation. A p -value 0.05 was considered statistically significant.

3.1 Cyanoacrylate embolization surgery and laboratory index

In the current study, the laboratory index of the patients who suffered from the venous disease is represented in Table 3. The participant’s blood samples were collected before surgery and two weeks after CEA application. There were no significant changes were observed before and after CEA surgery. EOS count was 0.15 ± 0.01 before CE ablation therapy and 0.19 ± 0.02 after the surgery. EOS counts may change when the body fighting an infection such as parasitic infection or allergic reaction [22, 23]. In our group, there were no significant changes. Also, other peripheral blood cell counts and biochemical parameters were not changed significantly. CA; In contact with blood and tissue, it turns into a binding membrane by polymerization, forming a strong tissue bond and provides occlusion of the incompetent vein. The best adhesion strength is reached after approximately 2.5 min. However, it has low fluidity and high flexibility. With its rapid initial polymerization National Institute [11], it significantly reduces the risk of deep vein embolization, and with its sufficient elasticity feature, it does not limit movement and increases patient comfort [18]. The use of CA for treating venous insufficiency is increasingly common. However, there are few studies on the effects of cyanoacrylate on the organism at the cellular level after treatment.

3.2 FT-IR spectroscopic studies

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Figure 1a illustrates FTIR spectra of human blood collected from patients before (black spectrum) and after (red spectrum) surgery. In these spectra peaks corresponding to functional group building proteins, lipids, carbohydrates, were marked. Peaks at 1082 cm - 1 and 1165 cm - 1 are co by the vibration of C–O and bending vibration of C–O–H [34]. Amide vibrations such as amide III, amide II, and amide I, were placed at 1238 cm - 1 and 1304 cm - 1 , 1531 cm - 1 , and 1635 cm - 1 wavenumbers, respectively [36]. The vibration of C = O is responsible for the peak at 1392 cm - 1 , while the peak at 1454 cm - 1 is originated from C–H stretching vibrations [37]. The peaks around 3278 cm - 1 are originated from O–H stretching vibrations. The vibration modes of peaks at 2958 cm - 1 , 2931 cm - 1 , and 2872 cm - 1 wavenumbers originate from the asymmetric vibration of C–H in CH 3 , asymmetric vibration of C–H in CH 2 , and symmetric vibration of C–H in CH 3 , respectively [38]. These functional groups in the structure of the lipid can be found. Obtained spectra showed that the analyzed two groups have differed from each other in the absorbance values. In the spectrum collected after surgery, the absorbance of peaks placed before 1700 cm - 1 had lower values, than the same peaks however in the sample collected before surgery. In the range corresponding to CH vibrations, the value of absorbance was similar in both groups. It was known, that the risk factors for chronic venous disease are: age, sex, but what is very important, when we used spectroscopic methods, which give information about chemical compositions, high weight, and cholesterol level [39, 40]. Consequently, the influence of weight and cholesterol in the FTIR spectra can be seen. Also, genetic factor plays a very important role in venous disease, therefore in Fig. 1b and c FTIR spectra in selected ranges are shown. From amide II and amide, I IR range, a change in the structure of proteins, can be noted when we compared samples before and after surgery, Fig. 1b. Moreover, in both selected IR regions, higher absorbance values in the samples collected before surgery were observed, (Fig. 1b and c) respectively. To show, which differences in the absorbance values were significant, one-way ANOVA followed by Tukey’s post hoc test was performed and the results were presented (see Fig. 2). Figure 2 showed that the average value of absorbance of peaks at 1165 cm - 1 , 1238 cm - 1 , 1304 cm - 1 , 2958 cm - 1 in the samples after surgery was higher than in the samples before surgery. However, in the case of peaks corresponding to amide III, amide II, amide I, and O-H vibrations, absorbance values were lower in samples after surgery. In the case of CH vibrations from lipids functional groups, the values of absorbance were very similar when we compared the analyzed group of samples. Continuing, differences in the value of absorbance were statistically significant only in amide II, amide I, and OH vibrations. This suggests, that amides vibrations could be used to differentiate blood samples collected from people suffering from venous disease, which had and had not surgery. Therefore, we decided to perform deconvolution of amide I vibrations, to show differences in the structure of the secondary protein caused by surgery (Fig. 3). When we compared two analyzed groups, different positions of individual secondary proteins structure were observed. Consequently, also the shape of fitted curves has differed from each other. Finally, statistically significant differences in the amount of α -helix and β -harmonica in both analyzed groups, were visible. In the blood collected before surgery, higher peaks area of α -helix and f β -harmonica were noticed in comparison with samples after surgery (see Fig. 3c). However, in both groups of samples, a higher amount of β -harmonica was visible.

FTIR spectra contain a lot of data, to be able to show, that using the FTIR technique it is possible to assess the blood of people suffering from venous disease, the PCA analysis was performed. The analysis performed for three different spectral ranges will show whether it is possible to differentiate by FTIR spectroscopy the blood of patients before and after surgery (Fig. 4). We decided to make PCA analysis for three separate IR regions: fingerprint, amide II with amide I, and region characteristic for CH lipids vibrations. Consequently, we choose the IR range of chemical compound, which the balance changes during venous disease [40].

PCA analysis of all analyzed IR ranges (Fig. 4a1–c1, respectively) showed, that it is possible to distinguish blood collected from patients suffering from venous disease, which was before and after surgery. For IR range 800 cm - 1 –1800 cm - 1 PC1 was significant in 79.9%, while PC2 – 19.4%. In the case of amide II and amide I region, the values of PC1 and PC2 were 88.7% and 10.6%, respectively, while in CH lipid vibrations range – 88.2% and 10.8%, respectively. Moreover, the Partial Least Square P-P plot of residual showed, that obtained from PCA analysis variances, had normally distributed.

To obtain information about which FTIR signals can be correlated with laboratory conditions results, the Pearson correlation test was performed (Table 4). In this section, only the correlation between analyzed features with the highest p-value will be described, while all p values for all analyzed factors were placed in Table 4. From correlation test shows that the value of WBC correlates with absorbance at 2858 cm-1 wavenumber. There are works, where authors showed a correlation between lipids and WBC levels [41]. Tolani et al. showed a significant relationship between elevated high-density lipoprotein (HDL) cholesterol levels and decreased monocyte numbers [42]. Other authors found significant positive associations between triglyceride levels and lymphocyte, neutrophil, monocyte, and basophil count as well as sex-dependent inverse relationships between HDL cholesterol levels and leukocyte counts [44]. Increased triglyceride and low-density lipoprotein (LDL) cholesterol levels were associated with increased total WBC, monocyte, lymphocyte, and neutrophil counts and increased levels are associated with increased lymphocyte number [44, 45]. Moreover, the correlation between NEU and OH vibrations, and between HCT and 1082 cm - 1 , were found. Furthermore, a high correlation PLT and 1165 cm - 1 , was noticed.

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Table 4

Pearson correlation test for a medical condition (blue marked) and FTIR results (yellow marked) with bold the highest p -value ( 0.9)