Comparison of Autologous Blood Coagulum (ABC) and Suture at Recurrence Rate and Graft Stability of Postoperative Primary Pterygium Using Conjunctival Autograft: A Meta-Analysis From Randomized Controlled Trial

Introduction: The main challenge of pterygium management is the postoperative recurrence rate. Currently, the use of surgical sutures for conjunctival autograft fixation, which has prolonged surgery duration, has a risk of increased inflammation and infection. Recently, some reports about autologous blood coagulum (ABC) for conjunctival autograft fixation. This technique has a minimal cost and minimal risk of infection. Purpose: The study aimed to evaluate the postoperative recurrence rate and graft stability after using ABC and sutures. Methods: A systematic research was done on Pubmed, Cochrane Library, and Science Direct online databases for all relevant randomized controlled trials (RCTs) up to 18 July 2020. The collected RCTs were independently screened and identified to match the inclusion criteria. The relevant data were compiled in population, intervention, control, outcomes (PICOs) format and analyzed with Review Manager 5.3 software. Results: Nine RCTs involving a total of 764 patients were assessed. The primary outcome indicates that ABC significantly reduced the recurrence rate compared to sutures (RR = 0.51, 95% CI 0.27 to 0.98, p = 0.04). Meanwhile, in graft stability, the suture was statistically better than ABC (RR = 1.95, 95% CI 1.27 to 3.01, p = 0.002). As a secondary outcome, the duration of surgery was significantly shorter in ABC than in sutures (MD -15.22, 95% CI -22.57 to -7.88, p<0.0001). Conclusions: Although the graft is less stable, ABC may be a promising approach after pterygium excision. The use of autologous blood for conjunctival autograft fixation in primary pterygium was associated with a lower recurrence rate and shorter duration of surgery.

Introduction beta rays, excimer lasers, argon lasers, thiotepa and antimetabolites, conjunctival autografts, and amniotic membrane transplantation. [7] Autologous conjunctival transplantation with or without limbal stem cells is a popular surgical treatment for pterygium because of its low recurrence rate and no complications. [8] The most commonly used method of autologous conjunctival fixation is a suture, however, it has disadvantages such as long operation time, infection, inflammation, suture abscess, and postoperative patient discomfort. [9] The latest autologous conjunctival fixation method is autologous blood coagulum (ABC). With this technique, the bleeding vessels are not coated, which allows a thin layer of blood to form on the sclera. The graft is then pressed against this thin layer, allowing the natural blood clot to hold. [10] The advantages of using ABC techniques are that the material is available to the patient, has the lowest cost, causes minimal infection, involves no risk of blood-related disease, and avoids complications associated with the use of sutures. Graft fixation using an ABC reduces patient discomfort and shortens operative time. [11] Ophthalmologists in various countries have developed randomized controlled trials (RCTs) studies of this method to evaluate its safety and efficacy. To determine the overall effectiveness of the existing RCTs, this study would like to summarize the RCT results regarding the effectiveness of the ABC on the conjunctival autograft technique in pterygium surgery compared to sutures in a meta-analysis study. This study evaluates the postoperative recurrence rate and graft stability after using ABC and sutures.

Data search method
This systematic review was based on the Cochrane Handbook for Systematic Reviews of Intervention guidelines and PRISMA guidelines. [12], [13] A computerbased data search was carried out independently by each author. Two investigators (AK and MP) performed the literature screening independently and determined the eligibility of the studies. Any disagreements between independent investigators were resolved by discussion and/or consultation with the senior investigator (DL).
We searched for relevant studies for ten years, from January 2010 to December 2020. We limit the study because the ABC studies have been relatively new in the last ten years. Using keywords with the relevant Medical Subject Headings (MeSH) format that consist combining of ("primary pterygium") or ("pterygium")) and (("conjunctival autograft") or ("autologous graft conjunctival") or ("conjunctival graft")) and (("autologous blood coagulum") or ("blood coagulum")) were inputted in search engines, including Pubmed, Cochrane Library, and ScienceDirect. We use Boolean operators to improve search results. Data pooling was done from June 2020 to July 2020.

Eligibility criteria
The inclusion criteria of this study were RCTs, with the study population consisting of male and female patients aged 18-75 years with primary pterygium who underwent pterygium excision procedures and reconstruction using conjunctival autograft with ABC fixation or suturing technique. The process of blood coagulum intervention in each study is shown in Table 1 Standards and variations of procedures for using an ABC are listed in Table 1-the postoperative recurrence rate and graft stability as primary outcomes and duration of surgery as secondary outcomes. Graft stability was asses in terms of the number of patients with graft retraction, displacement, dehiscence, or graft loss.

Sensitivity analysis
Sensitivity analysis in this study were performed by repeating the primary analysis with modified data sets or statistical methods to determine whether these changes

Author
Country Population The ABC method allowed the bare sclera to bleed freely for around 3-4 minutes. The conjunctival autograft is then removed, and the bare sclera is replaced. The surgeon waited around ten minutes before gently pressing the graft to promote fibrin production.

Intervention Control Process of ABC
Celik, 2018 Turkey Pterygium 15/15 Fixation with ABC Suture with 10/0 nylon For around 3-4 minutes, the bare sclera was allowed to bleed naturally. The conjunctival autograft is then removed, and the bare sclera is replaced. The surgeon waited around ten minutes for fibrin production and adhesion. Apply minimal pressure over the graft to assist in compliance.
Phani, 2017 India Pterygium 50/50 Fixation with ABC Suture with 8/0 nylon A thin layer of fresh blood must be applied over the bare sclera to give autologous fibrin. Hemostasis was allowed to occur spontaneously without using cautery to supply autologous fibrin for naturally attaching the conjunctival autograft in place without tension. The graft was fixed in place for ten minutes with mild pressure applied over the graft.

Study characteristics
The study selection process is shown in Figure 1. RCTs were used in the nine studies [7], [14], [15], [16], [17], [18], [19], [20], [21] that matched the inclusion criteria. Table 1 lists the characteristics of the nine studies. In total, 714 patients were included in this meta-analysis, 320 of whom were treated with ABC, and the remaining 394 were control populations with sutures.
All inclusion studies used conjunctival autograft after excision of pterygium. There were five studies in the control group using 10-0 nylon for fixation of the graft, three studies using 8-0 vicryl and one study using 8-0 nylon. All case groups used the patient's blood for conjunctival graft fixation, and almost all studies gave about ten minutes for hemostasis to occur. The process of using ABC from each study is further explained in Table 1.
The primary outcome of this study was the recurrence rate, as defined as fibrovascular growth extending

Risk of bias assessment
The risk of bias in each included study shown in Figure 2. All nine studies did randomization, but six studies [7], [14], [16], [17], [19], [20] have a low risk of bias in the "random sequence generation" parameter by elaborating the randomization method. Two studies [20], [21] did not explain the randomization method, and one study [18] had a high risk of bias assessment in this parameter.
Five studies [14], [16], [17], [19], [20] have a low risk of bias in the "allocation concealment" parameter. All of the included studies have an unclear risk of bias in the "blinding" parameters because the reviewers did not find the blinding statement in each study. The other reason is reviewers assume that the protocols of each study are difficult to blind; first, the participant must have informed consent so the intervention couldn't be blind to the participant; second, the surgery operator and the outcomes accessor could differentiate and find whether the participant had intervention method or control method quickly. So the reviewers think that blinding assessment in all studies is hard to apply. On the "incomplete outcome data'' parameter, eight studies [7], [14], [15], [16], [17], [18], [19], [20] have a low risk of bias, while on the "selective reporting" parameter, eight studies [7], [14], [15], [16], [17], [18], [19], [20] have a low risk of bias.
The risk of bias assessment of the included studies is summarized in Figure 3.

Sensitivity analysis
In sensitivity analysis, the recurrence results between ABC and suture were not statistically significant (Table 2).
Different from the outcome of graft stability, where there was a statistically significant difference between the ABC and suture. In the outcome of the duration of surgery,  there were also statistically significant differences between ABC and sutures. This sensitivity analysis was performed by excluding two studies [15], [21] from the initial primary analysis. Based on our study's bias analysis, both studies included low-quality bias.

Comparing ABC with sutures for assessing recurrence rate
From nine included trials, eight trials (644 participants, 644 pterygium operations) evaluated recurrence rates after surgery. A fixed-effect model is used due to significant homogeneity between studies (P = 0.88, I 2 = 0%). There is a statistically significant difference in the recurrence rate ( Figure 4) after primary pterygium surgery using the conjunctival autograft method between graft fixation using ABC and sutures (RR = 0.51, 95% CI 0.27 -0.98, p = 0.04), indicating that recurrence rate was lower in ABC than the suture. Egger's asymmetry test on the funnel plot represents no publication bias between studies (p = 0.8; p > 0.1) ( Figure 5 and Table 3).

Comparing ABC with sutures for assessing graft stability
From nine included trials, six trials (614 participants, 614 pterygium operations) evaluated graft stability after surgery. A fixed-effect model is used due to significant homogeneity between studies (P = 0.36, I 2 = 9%). There is a statistically significant difference in graft stability ( Figure   6) after primary pterygium surgery using the conjunctival autograft method between graft fixation using ABC and sutures (RR = 1.82, 95% CI 1.14 -2.90, p = 0.01), indicating that suture provided better graft stability than ABC.

Discussion
Nine studies found that the recurrence rate was significantly lower in ABC than in sutures. Based on the statistical analysis of this study, ABC has an efficacy value in reducing the risk of recurrence by 51% compared to sutures (the incidence of blood coagulum recurrence is ½ times lower than sutures). This finding is different from the meta-analysis by Zein et al. [22] where from these, studies, the recurrence rate did not differ significantly between blood coagulum and suture. This could be because the number of studies that were used was less.
Zein et al. [22] used four studies from 2013 to 2015; and this study used nine references from 2015 to 2020. The more studies included as material for analysis, the more valid the analysis results; on the other hand, the newer study will also increase the study's validity. The procedure of ABC was bare sclera allowed to bleed spontaneously for about 3-4 minutes. Then conjunctival autograft was replaced with the bare sclera. The surgeon waited about ten minutes by applying gentle pressure over the graft to adhere to fibrin formation.
Recurrence following successful excision presents a concern in pterygium surgery. In a research conducted by Huda and Khaleque [14] , using the ABC technique can minimize the recurrences; not one pterygium recurred after three months of surgery. The occurrence of recurrences in the pterygium is multifactorial.
Numerous studies have demonstrated that postoperative inflammation promotes the proliferation of blood vessel cells and fibroblasts, as well as subconjunctival fibroblast tissue, and the overexpression of matrix metalloproteinases leads to pterygium invasion. [23] The lower recurrence rate with the ABC is due to decreased postoperative inflammation. [24] Moreover, using silk or nylon sutures might lead to inflammation and migration of Langerhans cells to the cornea.
In the study conducted by Wit et al. [25] , there were no recurrences in 15 eyes at a mean follow-up of 9.2 months. Using an ABC produces pressure over the entire graft surface. There is no direct pressure on the edges of the graft, which minimizes scar formation on the subconjunctival. The presence of sutures can cause a long healing process and fibrosis. [25] In terms of graft stability, statistical analysis showed a significant difference between the ABC and the suture, where the suture provided better graft stability than the ABC. In this study, graft stability was a combination of the incidence of graft retraction, graft displacement, and graft loss. In the meta-analysis by Zein et al. (2018) [22] , not combining these parameters and separating, graft retraction and graft displace lower with sutures than the coagulum blood. Although this study combines one parameter, namely graft stability, the statistical analysis results are the same as those of Zein et al. [22] , where the stability of the graft is more stable in the suture than in the blood coagulum.
In the study of Singh et al. [2] , graft displacement cases were higher in the group with ABC (7.1%) compared to the suture group. It is thought that a graft displacement may occur when the patient rubs the eye operated on inappropriately due to a foreign body sensation.  This is supported by Zein et al. [22] , which state that displacement. [26] The disadvantage of having an ABC is challenging to manage the amount of blood in the sub graft; when more blood flows, a large clot is expected to form under the graft, and the contractions are caused by the clot results in the graft being released. [11] In terms of duration of surgery, statistical analysis showed a significant difference between the use of blood coagulum and suture, where coagulum blood provided a shorter duration of surgery than the suture. This is also to a meta-analysis study by Zeng et al. [22] , where the blood coagulum provides a shorter duration of surgery.
Suture-based graft fixation involves additional surgical procedures and a more extended period of operation, whereas autologous blood coagulum fixation is generally quick and straightforward. Surgery is performed in less time, leading to cheaper operating expenses, more patient satisfaction, and a decreased risk of infection.
However, The data analysis on autologous blood and sutures showed a high degree of heterogeneity. One explanation is that surgeons' abilities differ. Additionally, the duration of surgery was defined inconsistently between studies. [6] In addition, the method of each  Table 3. Egger's test from the funnel plot (regression test for funnel plot asymmetry).  journal does not blind the participants, personnel, and outcomes, so it is difficult for us to take the risk of biased parameters. Suggestions in this study were conducted in a multicenter RCTs study to get results with a larger population. Also made guidelines regarding ABC to have a uniform technique.

Conclusions
In conclusion, although the graft is less stable, autologous blood coagulum may be a promising approach after pterygium excision. The use of autologous blood for conjunctival autograft fixation in primary pterygium was associated with a lower recurrence rate and shorter duration of surgery.