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

Abstract

BACKGROUND:

The venous disease of the legs is a common disease among adults that may lead to a deterioration in the structure and concentration of biomolecules. N-Butyl Cyanoacrylate Ablation Surgery (NBCA) or cyanoacrylate embolization (CAE) technique to adhesive the saphenous vein is an alternative method for the treatment of venous disease.

OBJECTIVE:

We aimed to show what kind of changes occurs after CAE surgery using FTIR spectroscopy combined chemometrics. We compared before and after surgery blood sera of patients to find whether a correlation between spectral data and laboratory indexes. We studied the blood sera of those who suffered from varicose veins and treated them by CAE technique.

METHODS:

In order to examine the molecular profiles in blood sera who underwent the CAE technique of the great saphenous vein for the treatment we used Fourier Transform InfraRed spectroscopy (FTIR) spectroscopy of blood samples of patients before and after surgery as a fast diagnostic technique. To obtain information about the spectra variation among the types of samples Principal component analysis (PCA) was performed for fingerprint, amide II with amide I regions. To find normality among variations Partial Least Square P-P plot of residual was performed.

RESULTS:

Absorbance values were statistically significant only in amide II, amide I, and OH vibrations. In the blood collected before surgery, higher peaks area of $\alpha$ -helix and $\beta$ -harmonica were noticed. However, in both groups of samples, a higher amount of $\beta$ -harmonica was visible. Pearson correlation analysis showed that the value of white blood cells (WBC) correlate with absorbance at 2858 cm ${}^{-1}$ wavenumber. Moreover, a correlation between neutrophil (NEU) and OH vibrations, and between hematocrit (HCT) and 1082 cm ${}^{-1}$ , were found. Furthermore, a high correlation Platelets (PLT) and FTIR peak at 1165 cm ${}^{-1}$ , was noticed.

CONCLUSIONS:

This methodology suggests with PCA analysis CAE caused structural and quantitative chemical changes in blood samples of patients.

Keywords

Varicose veins Cyanoacrylate Ablation Surgery blood parameters Fourier Transform InfraRed spectroscopy Principal component analysis

1. Introduction

The venous disease of the legs affects is a common disease affecting the adult population, especially more usual in women (25%) than in men (15%) [1]. The occurrence of varicose veins depends on many personal factors, including gender, age, pregnancy, posture at work, obesity, genetics, and lifestyle [2]. Venous insufficiency can impair the quality of life of the person due to the complaints of pain and swelling in the leg and can progress with thrombosis and even ulcerations if left untreated [3, 4]. Color Doppler Ultrasonography (CDUS) is the most used technique in the diagnosis and follow-up of venous insufficiency [5, 6]. With this imaging method, detailed information about the condition of the veins, such as the diameter and structure of the vein, abnormal vascular structures, and the flow profile of the blood may be obtained [7]. Stripping and ligation, which have been used for many years and are classical surgical techniques, have started to be replaced by less invasive methods recently. These new techniques used in venous insufficiency are laser ablation (EVLA) sclerotherapy, radiofrequency ablation (RFA) [8], and Cyanoacrylate Ablation Surgery or cyanoacrylate embolization (NBCA or CAE) [9]. As the use of these minimally invasive techniques has become widespread, their effectiveness is better understood [10]. Compared to conventional surgical methods, it is as effective as surgery for treating varicose veins. In addition to the high success rates of these new methods, the contribution to the comfort of the patient is great, the patient is discharged quickly, the incidence of complications decreases, and less pain occurs [9]. Among these techniques, CAE techniques are used endo venous for treating venous insufficiency and varicose veins for biochemical ablation. The diagnosis of chronic varicose veins and venous insufficiency is usually decided clinically [2, 3]. It initially consists of history and physical examination. Doppler ultrasound imaging is used for clinical confirmation of the disease. The Clinical-Etiology-Anatomy-Pathophysiology (CEAP; Table 1) scoring system is important to classify the clinical severity of venous disease and to identify the contributing pathology [1, 2, 8]. Cyanoacrylate (CA) is a substance used in the CAE technique to adhesive the saphenous vein [10]. Although it has been known since the 1960s, its use to treat venous insufficiency is relatively new compared to other minimally invasive treatments [9]. Recently, articles about the use of cyanoacrylate in this direction have started to occur in the literature. It has been observed that it is an effective treatment method by providing occlusion between 92% and up [11, 12]. Cyanoacrylic adhesives used in this treatment are a chemical family with strong and fast adhesion properties. The CA family is named for alkyl groups that are variable with cyano and ethylene groups. N-butyl cyanoacrylate was used until 1970, but it was not allowed to be used more by the US Food and Drug Administration (FDA) due to irritation on the skin [13]. While N-butyl cyanoacrylate itself has an irritative effect, today different cyanoacrylates formed by adding more compounds to CAs are easily used in the medical field [14]. The use of CA for treating venous insufficiency is becoming increasingly common and studies on this subject have recently started to be published. However, studies on the effects of cyanoacrylate at the cellular level in the organism after treatment are very few [15]. Despite promising early outcomes, adverse reactions to CA have been recognized. The first in a human study of reported the development of phlebitis requiring oral nonsteroidal anti-inflammatory drug treatment in 6 patients [14]. In thirty-four patients treated with NBCA, reporting development of an abnormal skin reaction described as erythema, itchiness, edema, pain, and tenderness over the treated area, all of them recovering fully within 2 weeks [16]. Jones et al. reported a case in which, after treatment with NBCA, worsening leg pain and erythema developed 13 days post-operatively despite treatment with oral/topical analgesic. The patient had a positive patch test response for CA and continued to experience erythema, leg pain, and swelling up to 4 months post-operatively. Decided to have the affected vein endoscopically excised. The excised vein was histologically examined and seen that hypersensitivity reaction [17]. In some animal experiments (rabbits and pigs), histopathological changes such as acute inflammation of the vascular endothelium were observed after NBCA administration. It has been shown that acute inflammatory effects are seen without proliferation of elastic fibers two weeks after this application, followed by a chronic granulomatous foreign body reaction within two months [18, 19]. Fiengo et al. reported that a 45-year-old female patient showed a hypersensitivity reaction after NBCA application. Nine days after the NBCA application, the patient observed swelling and itching on the kneecap and erythema. These symptoms disappeared after 3 days of antihistamine administration. About 20 days after surgery, the patient developed more erythema around the groin, followed by a rash on the arms, abdomen, chest, neck, shoulders, and scalp. Some important latest study informations are given in Table 2. The results of the tests showed that this hypersensitivity formation was caused by cyoacrylate [20]. In the current study, we aimed to examine the effect of n-butyl cyanoacrylate ablation in varicose vein surgery in patients before and after surgery. For this purpose, we obtained blood samples of patients who underwent the CAE technique of the great saphenous vein for the treatment. Afterward, the molecular profiles in blood sera were examined using FTIR spectroscopy, which is a fast, convenient analytical technique that can ensure knowledge about biomolecules of the human blood samples. Bringing new evidence with these comparisons and perspectives, the changes in the blood before and after the operation using both bioclinical, biochemical and spectroscopic data would suggest a multi-disciplinary knowledge using different methodologies. Another novelty point is to assess the effect of the biogradable material cyanoacrylate to make a new interpretation of known material that is a used for venous ablation in the surgery. Our study suggests to add new knowledge to the current pool of technology and health sciences pool of knowledge.

Table 1
CEAP classification for venous disorders in clinical diagnosis [1, 8]

Clinical classification (C)
C0	No visible or palpable signs of venous disease
C1	Telangiectasias or reticular veins
C2	Varicose veins
C3	Swollen ankle (edema)
C4a	Pigmentation or eczema
C4b	Lipo dermatosclerosis or atrophie blanche
C5	Healed venous ulcer
C6	Active venous ulcer
Etiologic classification (E)
Ec	Congenital
Ep	Primary
Es	Secondary
Anatomic classification (A)
As	Superficial
Ad	Deep
Ap	Perforator
Pathophysiologic classification (P)
Pr	Reflux
Po	Obstruction

Table 2

Some important latest study information about venous ablation techniques

Author	Publication name	Model	Year	Techniques	Results
Jones et al. [17]	Cyanoacrylate adhesive for the closure of truncal veins: 60-day swine model results	The venaseal (medtronic, minneapolis, minn) cyanoacrylate closure system	2019	Nonthermal technique for ablating saphenous veins using a proprietary n-butyl cyanoacrylate	The patient elected to have the vein excised, and the histologic features were consistent with a type $\imath$ v hypersensitivity reactio
Parsi et al. [20]	Granuloma formation following cyanoacrylate glue injection in peripheral veins and arteriovenous malformation	Cyanoacrylate adhesive closure	2020	Great saphenous vein were injected with venablock ${}^{\text{TM}}$ glue under ultrasound guidance	After the use of n-butyl cyanoacrylate, extravascular foreign body cavity granulomas appeared with fibrosis and lymphoid aggregates
Almeida et al. [18]	Cyanoacrylate adhesive for the closure of truncal veins: 60-day swine model results	Animal model	2011	A sheath was positioned to the junction of the superficial epigastric and abdominus rectus veins in 2 swine, and ultrasound-guided injection of ca glue	Histopathological changes such as acute inflammation of the vascular endothelium and segmental wall thickening, and fibrosis were observed

2. Materials and methods

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 $\geqslant$ 35 years and $\leqslant$ 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.

Table 3
Laboratory indexes of patients suffering from a varicose vein before and after CEA surgery

	Before surgery mean (SE)		After surgery mean (SE)		Reference (unit)
Peripheral blood cells
Lymphocytes (LYM)	2.34	$\pm$ 0.15	2.49	$\pm$ 0.13	1.3–3.5 (10 ${}^{3}$ $\mu$ L)
Neutrophils (NEU)	4.88	$\pm$ 0.43	5.21	$\pm$ 0.46	2.1–6.1 (10 ${}^{3}$ $\mu$ L)
White blood cells (WBC)	8.03	$\pm$ 0.59	8.59	$\pm$ 0.58	4.3–10.3 (10 ${}^{3}$ $\mu$ L)
Monocytes (MONO)	0.6	$\pm$ 0.1	0.6	$\pm$ 0.1	0.3–0.9 (10 ${}^{3}$ $\mu$ L)
Eosinophils (EOS)	0.2	$\pm$ 0.0	0.2	$\pm$ 0.0	0–0.5 (10 ${}^{3}$ $\mu$ L)
Red blood cell counts (RBC)	4.7	$\pm$ 0.1	4.7	$\pm$ 0.1	4.2–5.4 ( $\mu$ L*L)
Basophils (BASO)	0.1	$\pm$ 0.8	0.1	$\pm$ 0.1	0–0.2 (10 ${}^{3}$ $\mu$ L)
Platelets counts (PLT)	253.4	$\pm$ 10.0	256.8	$\pm$ 12.2	156–373 (10 ${}^{3}$ $\mu$ L)
Biochemical parameters
AST	24.6	$\pm$ 5.8	21.1	$\pm$ 1.7	15–42 (IU/L)
ALT	20.1	$\pm$ 2.1	21.3	$\pm$ 2.9	10–40 (U/L)
CRP	5.0	$\pm$ 0.8	4.2	$\pm$ 0.6	$<$ 3.00 (mg/L)
HGB	13.4	$\pm$ 0.3	13.4	$\pm$ 0.3	13–16 g/dl*L
HCT	40.1	$\pm$ 0.7	40.1	$\pm$ 0.7	36–48 %*L
Creatinine	0.7	$\pm$ 0.1	0.8	$\pm$ 0.1	0.5–1.4 mg/dL
Urea	28.1	$\pm$ 1.8	30.5	$\pm$ 1.4	10–40 mg/dL

2.3 Endo venous application of cyanoacrylate embolization surgery

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 ${}^{\circ}$ 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 $\mu$ 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 $\pm$ 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 $p<$ 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.

2.5.1 Principle component analysis (PCA)

We performed Principal component analysis to obtain information about the spectra changes between the sample’s types. PCA reduced the dimensionality, the number of variables of the data while maintaining as much variance as possible [29, 30]. It can be used to evaluate complex spectra which may consist of hundreds of absorbance values by reducing each spectrum to a single point in a lower-dimensional space in an approximate decomposition of the data matrix $X_{\textit{mxn}}$ containing m samples and n features, a matrix with a lower rank h is formed, for which h $\leqslant$ n is establishing. An IR spectrum is measured by its absorption values at different wavelengths. PCs are the new latent variables h that are uncorrelated. The PCs combine the original data and are oriented to produce the greatest variance. In the simplest terms, PCA is an unsupervised method that is not capable of classifying and identifying different samples. The distinct differences that are found in the data can be shown clearly through PCA. This fact can be sourced [31, 32].

2.5.2 Partial least square analysis (PLS)

Partial least squares a regression method and a multivariate statistical technique that allows comparison between multiple response variables and multiple explanatory variables [33]. Furthermore, considering the small number of samples in this work, a partial least squares P-P plot of residuals was implemented. When the number of predictors is much larger than the number of samples in the high or wide data set, it is used for multicollinearity problems related to complex biological data, such as this study. Furthermore, in this study we present diagnostic plots as a normal probability plots. This plot show, that the data is approximately linear supporting the condition that the error terms are normally distributed, which means that also discriminant analyses can be performed.

The objective function in PLS analysis to be solved can be written as:

$\displaystyle\textit{Arg}_{u^{\prime}_{h}u_{h}=1,v^{\prime}_{h}v_{h}=1}\max?% \quad\textit{cov}^{2}(v^{\prime}_{h}X,v^{\prime}_{h}Y).$

where $u_{h}$ , $v_{h}$ mean the $h$ th left and right singular vector, which are called also as loading vectors and are associated to the $X$ and $Y$ data set respectively [34].

3. Results and discussion

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 $\pm$ 0.01 before CE ablation therapy and 0.19 $\pm$ 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

Figure 1.

FTIR spectra of blood collected before surgery (black spectrum) and after surgery (red spectrum) (a); IR range of amide II and amide I vibrations (b); IR range of CH vibrations from lipids (c).

Figure 2.

Mean $\pm$ SEM intensity values of the individual peaks for two analyzed groups. Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Data were considered as significant when ${}^{*}p<$ 0.05 vs. before surgery (black).

Figure 3.

Curve-fitting analysis of the amide I profile of blood collected from patients before (a) and after (b) surgery. Mean $\pm$ SEM intensity values of the individual peaks (c) corresponding to $\alpha$ -helix and f $\beta$ -harmonica for groups of patients before surgery (black) and after surgery (red).

Figure 4.

PCA analysis of blood collected from patients suffering from venous disease, which was before (black dot) and after (red dot) surgery. PCA analysis for IR ranges: 800 cm ${}^{-1}$ –1800 cm ${}^{-1}$ (a1); 1500 cm ${}^{-1}$ –1700 cm ${}^{-1}$ (b1) and 2800 cm ${}^{-1}$ –3000 cm ${}^{-1}$ (c1) with loadings plots of PC1 (a2–c2), PC2 (a3–c3) and Partial Least Square P-P plot of residual of obtained results (a4–c4).

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 $\alpha$ -helix and $\beta$ -harmonica in both analyzed groups, were visible. In the blood collected before surgery, higher peaks area of $\alpha$ -helix and f $\beta$ -harmonica were noticed in comparison with samples after surgery (see Fig. 3c). However, in both groups of samples, a higher amount of $\beta$ -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.

Table 4

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

Table 4, continued

4. Conclusion

In this study, the objective was to examine the possible changes in the blood samples taken before and after the operation of the chemical substance (CA) used to close the saphenous vein with the FTIR spectroscopic method. By detecting the changes and molecular compositions at the molecular level, it is aimed to determine possible changes in the secondary structure of proteins and to obtain a metabolic profile. Thus, it is aimed to obtain information about the course of treatment and the effect of the chemical used. In summary, for the first time, the present study revealed several profound contextual, structural, and functional changes in biomolecules such as proteins and lipids of patients who suffered from C2 to C4 grade venous insufficiency who treated by endo-venous cyanoacrylate. We used infrared spectroscopy to investigate variations. Additional infrared coupled chemometric analysis such as PCA, PLS, Curve-fitting, and correlation analysis enabled us to discriminate before and after surgery group as a new approach. Our results presented, PCA of all analyzed IR range showed, that it is possible to distinguish blood collected from patients suffering from venous disease, before and after surgery in the case of amide II, amide I region, and CH lipid vibrations range in different sensitivity and accuracy. Hence, FTIR spectroscopy and curve-fitting analysis of amide I profile successfully applied to identify qualitative and quantitative changes of protein structure in blood sera of the groups. Interestingly, in the range corresponding to CH vibrations, the value of absorbance was similar in both groups because of risk factors known for chronic venous disease. Another novelty of our study is to find a correlation between laboratory indexes of peripheral blood cell counts and spectroscopic data. It is verified that venous insufficiency blood sample parameters such as WBC, NEU, and PLT have absorbance in the wavenumber of FTIR spectroscopy in addition to changes in biomolecules.

Based on the data we have obtained as a result of this study, future studies are planned. Among the studies planned to be carried out, primarily oxidant/antioxidant enzymes and involved in these enzymes structure in quantities of the elements will be investigated. Considering these data, it is aimed to discuss whether the changes that may occur are due to the cyanoacrylate used for ablation. It is planned to investigate the effectiveness of such chemical substances and to examine the health effects of these substances and to present new suggestions.

Data availability statement

The raw/processed data employed to obtain these findings are available from the corresponding author upon reasonable request.

Funding

This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Footnotes

Conflict of interest

The authors declare that there are no known competing interests.

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

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

Table 1 CEAP classification for venous disorders in clinical diagnosis [1, 8]

2.1 Ethics

2.2 Study design

Table 3 Laboratory indexes of patients suffering from a varicose vein before and after CEA surgery

2.4 Sample preparation for the acquisition of ATR-FTIR spectra

2.5 Statistical analyses for FT-IR spectra

2.5.1 Principle component analysis (PCA)

2.5.2 Partial least square analysis (PLS)

3. Results and discussion

3.1 Cyanoacrylate embolization surgery and laboratory index

3.2 FT-IR spectroscopic studies

Data availability statement

Funding

Footnotes

Conflict of interest

References

Table 1
CEAP classification for venous disorders in clinical diagnosis [1, 8]

Table 3
Laboratory indexes of patients suffering from a varicose vein before and after CEA surgery