A Study of Blood Viscosity and Inflammatory Biomarkers’ Levels in Bilateral Primary Varicose Veins/Reticular Veins as Predictive Markers

INTRODUCTION

Varicose veins are the most common form of chronic venous diseases of the lower limbs, affecting up to 30% of the population with a slightly higher prevalence in women than in men (25–33% and 10–20%, respectively).^[1–4] Incidence of varicose veins in the adult population has been shown to vary among populations (between 10% and 60%) and to increase with age in various studies.^[5–10] Enhanced oxidative stress has been implicated as the causative mechanism behind vessel wall injury in recent studies.^[11] Reactive oxygen species (ROS) are involved in the blood coagulation cascade.^[12] Experimental data have demonstrated that ROS can modify the fibrinolytic response, affecting the production of tissue Plasminogen Activator (tPA) and its inhibitor Plasminogen Activator Inhibitor-1 (PAI-1), increasing PAI-1 expression by endothelial cells.^[13–15] Several hematological abnormalities, such as increased blood cell count and aggregation, increased low-shear rate viscosity, and elevation in plasma fibrinogen, have been identified in venous blood samples collected from the feet of patients with chronic venous diseases.^[16,17] Therefore, this study aims to assess the biomarkers of inflammation and blood viscosity as predictive markers in patients with primary bilateral varicose veins or reticular veins that might progress to varicose veins.

MATERIAL AND METHODS

The cross-sectional study with a sample size of 40 patients from 9 different occupations was conducted in the Post Graduate Department of General Surgery, Moti Lal Nehru (MLN) Medical College, Prayagraj, for a period of 1 year from October 2021 to September 2022. The Institutional Ethics Committee (IEC) of MLN Medical College, Prayagraj, has approved this study; approval reference number is - (ECR/922/inst/UP/2017). Patients, including policemen, students, farmers, teachers, surgeons, housewives, army officers, tea stall owners, and guards, aged between 18 and 65 years and admitted to the unit with bilateral primary varicose veins/reticular veins falling under reticular veins and symptomatic varicose veins (C1, 2s), primary etiology (Ep), superficial veins (As), reflux pathology (Pr) categories of Clinical, Etiologic, Anatomic, Patho-physiologic (CEAP) classification classifying chronic venous diseases with the exception of those with telangiectatic veins, were included in the study.^[18] This includes patients with clinically present symptomatic bilateral reticular veins and varicose veins of primary etiology in superficial venous system with reflux pathophysiology. Blood samples were taken from the involved varicose veins in the study group and in the control group and sent for evaluation, and blood viscosity was measured using Capillary Viscometer. Evaluated inflammatory markers were:

• tPA

• PAI-1

• Fibrinogen

Serum tPA levels (ng/mL), serum PAI-1 levels (ng/mL) and serum fibrinogen levels (mg/dL) were measured using quantitative enzyme-linked immunosorbent assay (ELISA), which followed the Sandwich-ELISA principle. The concentration of inflammatory biomarkers in the samples was calculated by comparing the optical density (OD) of the samples to the standard curve. Blood viscosity of venous blood samples was measured using Capillary Viscometer based on Poiseuille’s Law using the following formula:

where ρ is the blood density, t is the time of outflow of the sample, ρ₀ and t₀ are density and the time of outflow of the reference liquid (water) respectively. The final analysis was done with the help of Microsoft EXCEL and Statistical Package for the Social Sciences (SPSS) software. For statistical significance, p-value less than 0.05 was considered.

RESULTS

The control group included 20 participants with CEAP grade C0. Study group of 20 participants was divided into the C1 study group, which included 6 patients who had C1, Ep, As and Pr with bilateral reticular veins, and the C2 study group, which included 14 patients who had symptomatic bilateral varicose veins (C2s, Ep, As, and Pr) [Figure 1].

• The highest number of cases was in the age group of 31–40 years [Figure 2].

  

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  Figure 1:

  Distribution of cases vs. control; C0: Control group, C1: Study group, C2: Study group.

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  Figure 2:

  Distribution of patients according to age intervals.

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• Mean age of male and female patients was 38.67 ± 13.31 and 33.3 ± 6.88 years, respectively.

• Mean ages of male patients in the study group and control group were 40 ± 15.65 and 37.5 ± 11.28 years, respectively, while that of female patient in the study group, and the control group were 30.67 ± 5.79 and 37.25 ± 7.18 years, respectively.

• The male–female ratio was 3:1. The ratio in the study group was 7:3 and the ratio in the control group was 4:1. Both the groups contained mainly male patients for the study (70% in the study group and 80% in the control group).

DISCUSSION

In the present study, the mean age of patients with bilateral (B/L) varicose veins, reticular veins, and the control group was 35.14 ± 11.9, 42 ± 18.33 and 37.33 ± 12.18 years, respectively. Maximum patients belonged to the age group of 31–40 years. The difference between the different groups and the age of patients was statistically insignificant in contrast with Shafiuddin M et al. (2017) where most of the patients belonged to the age group of 21–30 years.^[19] The incidence of disease was found to be more in males as compared to females in B/L reticular veins patients and in B/L varicose veins patients. The lower incidence in females was probably due to less cosmetic concern in the middle and lower-class strata.

The relationship between occupation and different study groups was statistically insignificant. However, an increasing trend of mean value of standing hours was the lowest in the control group, followed by B/L reticular veins patients, and the highest in patients of B/L varicose veins. The relationship between the mean duration of standing hours in different study groups is statistically significant. In pairwise group comparisons, B/L varicose veins versus control group and B/L reticular veins versus control group, both groups have shown statistically significant difference. The difference between patients of B/L reticular veins and B/L varicose veins was statistically insignificant. Hence, the result that prolonged standing hours are associated with the development of B/L reticular or varicose veins irrespective of the occupation is in line with Shafiuddin M et al. (2017), Mirji et al. (2011) and Nagraj et al. (2014).^[19–21]

The highest mean blood viscosity values are found in patients of B/L reticular veins, followed by patients of B/L varicose veins and the control group. The relation between different study groups and blood viscosity was statistically insignificant. The results are in sync with Flore et al. (2015)^[22] and contrary to Androulakis & Panoysis et al. (1989).^[23]

The mean serum tPA values were in patients of B/L varicose veins, followed by the control group. The lowest mean value of serum tPA was seen in patients of B/L reticular veins. The relation between different study groups and serum tPA levels was statistically insignificant and in line with Shireman et al. (1996)^[8] and Yasim & Kilinç et al. (2007)^[24] and in contrast with Blomgren et al. (2001).^[15]

The mean value of serum PAI-1 in patients of B/L reticular veins was followed by patients of B/L varicose veins. The lowest mean value of serum PAI-1 was seen in the control group. The relationship between mean serum PAI-1 values, and different study groups was statistically insignificant and consistent with Shireman et al. (1996)^[8] and in contrast to Blomgren et al. (2001).^[15]

There is an increasing trend of mean value of serum fibrinogen levels with the lowest value in the control group followed by B/L reticular veins patients, and the highest value in patients of B/L varicose veins. The relationship between the mean serum fibrinogen levels in different study groups is statistically significant. In pairwise group comparisons, B/L varicose veins versus the control group, and B/L varicose veins versus B/L reticular veins, both groups have shown statistically significant difference. The difference between patients of B/L reticular veins and the control group was statistically insignificant. Hence, it is concluded that raised fibrinogen levels are associated with the development of B/L varicose veins, and not with B/L reticular veins. The results are consistent with Pola et al. (1994),^[17] Flore et al. (2015),^[22] Karahan et al. (2016),^[25] Tiwary & Kumar et al. (2020)^[26] and contrary to Yasim & Kilinç et al. (2007)^[24] and Gomez et al. (2012).^[27]

LIMITATIONS OF THE STUDY

• Due to time constraints and the limited availability of specific patients during the study period, the sample size was small and the results cannot be generalized to the whole population and so a predictive range of fibrinogen for the general population cannot be decided upon. Therefore, further extensive study with a large sample size is required to generate standard cut-offs.

• The results cannot be extended to cases of unilateral (U/L) primary varicose veins.

• The number of male patients was significantly higher than female cases. Therefore, gender bias, if any, has not been fully accounted for.

CONCLUSION

On the basis of our study, we conclude that blood viscosity and biomarkers of inflammation, that is, tPA and PAI-1 cannot be used as predictive markers in patients with bilateral primary varicose veins/reticular veins. Serum fibrinogen levels cannot be used as predictive markers for B/L reticular veins but they can be used as predictive markers for the development of B/L primary varicose veins and in our particular study with a predictive range of 189.4–327.9 mg/dL. The study also concludes that prolonged standing, irrespective of the occupation of the patient, is associated with the development of B/L reticular veins as well as B/L varicose veins.