TITLE: 
Autologous Cryoprecipitate for Attaching Conjunctival Autografts after Pterygium Excision



ABSTRACT.PURPOSE:
To report the efficacy, safety, and reliability of autologous cryoprecipitate in pterygium excision surgery and to compare it with the traditional method of using absorbable sutures, in regard to surgical time and the patient comfort.

ABSTRACT.MATERIALS AND METHODS:
A prospective interventional clinical study was carried out in a specialized eye clinic. A total of 54 patients (90 eyes) underwent surgical excision of the nasal pterygium (whether primary or recurrent) with conjunctival autograft obtained from the same eye. Patients were divided into two groups. Autologous cryoprecipitate was used in 47 eyes (glue group), and absorbable sutures (8/0 vicryl) were used in 43 eyes (suture group) to attach the free conjunctival graft. There were 42 primary and 48 recurrent nasal pterygia that were included in the study. The surgical time was noted, and post-operative pain was graded. Follow-up period ranged from 6 months to 18 months (mean 12 months). P < 0.05 was statistically significant.

ABSTRACT.RESULTS:
The medians of the visual analogue scale values were significantly lower in the glue group (P < 0.05). The median surgical time was statistically significantly lower at 11 min (range 9 min to 15 min) in the glue group, compared to 21 min (range 12 min to 28 min) for the suture group (P < 0.05). No significant intraoperative or post-operative complications were noted. Recurrence rate was 12%, and all recurrence cases occurred in the sutures group.

ABSTRACT.CONCLUSIONS:
Application of autologous cryoprecipitate glue instead of sutures for attaching the free conjunctival graft in pterygium surgery resulted in less post-operative pain and shorter surgical time. Additionally, there were no cases of recurrence during the follow-up in patients who received autologous cryoprecipitate glue during pterygium surgery.

BODY.INTRODUCTION:
Surgical management is the ultimate solution for progressive pterygia. The main challenge is to minimize the risk of recurrence. Multiple different procedures have been advocated in the treatment of pterygium. These procedures range from basic excision to sliding flaps or rotation of the conjunctiva, with and without adjunctive external beta radiation, excimer laser and/or the use of topical chemotherapeutic agents such as Mitomycin C. Because of serious complications of the adjunctive procedures,123456 most corneal surgeons prefer to use a free conjunctival graft with or without limbal tissue, after completely excising the lesion. Conjunctival autografting is safe and helps reduce the risk of recurrent pterygium.789101112
Traditionally a conjunctival graft has been attached to the underlying sclera with sutures. Recently, the use of fibrin glue rather than sutures has become popular. Fibrin glue has been adopted in ocular surgery to seal conjunctival wounds, to attach autografts in pterygium surgery, and free skin transplants in lid surgery, to repair injured canaliculi, and to glue minor corneal perforations and incisions in cataract surgery.131415
Previously, we have prepared natural fibrin glue (biological adhesive) from the patient's autologous plasma, for application in sutureless epikeratophakia. A preparation procedure for cryoprecipitate has been published for fibrin glue.161718 We believe that the reduction of the risk of blood-borne infections with the autologous product is a significant advantage.19
In this paper, we report the outcomes of surgical time and the long-term outcomes of patient comfort and recurrence rate, with the use of sutureless conjunctival autografts compared to sutured autografts in pterygium excision surgery, in a large group of patients.

BODY.MATERIALS AND METHODS:
We designed a prospective interventional clinical study to report the long-term outcomes of sutureless pterygium surgery for the primary and recurrent pterygium, and to compare them to conventional pterygium surgery (where sutures were used to attach the conjunctival autograft). Clinical data collections were undertaken following ethical approval and in accordance with the Declaration of Helsinki.
All surgeries were performed between December 2006 and June 2008 at a specialized eye clinic in Damascus-Syria. All patients underwent a full eye examination. They had either primary pterygium or recurrent pterygium that was symptomatic and clinically measured more than 2 mm from the limbus. Unusual or non-nasal pterygium cases were excluded.
All patients consented to study after being informed of the study nature and plan, and were provided with a leaflet that demonstrated the study plan and answered the frequently asked questions. Fifty-four patients (90 eyes) were given the option of whether to undergo pterygium excision with conjunctival autograft glued to the bed, or pterygium excision with conjunctival autograft sutured to the eye. All surgeries were performed by one surgeon.
Success was defined as no recurrent pterygium 6 months after surgery. Recurrence was defined as any fibrovascular regrowth across the limbus. Patients were asked to fill out a questionnaire that included the visual analogue scale (VAS) assessment and were requested to report the degree of pain at the end of every day for the 1st post-operative week. The VAS was used to evaluate postoperative pain. Patients were taught how to use the VAS; it is a basic scale that consists of a 10 cm line anchored at one end by a label “no pain” and at the other end by “worst possible pain”, and on the back, the scale is transformed to the numerical values from 0 to 100 for statistical analysis. As a reference point, we tested patients by installing one drop of Oxybuprocaine HCL 0.4% (Medicain-Medico Labs-Syria) and asking the patient to mark the severity of pain on the ruler. At a later stage, this value was subtracted from the post-operative VAS values. None of the patients received a systemic or topical analgesic post-operatively.
The preparation of the adhesive fibrin glue (fibrinogen) has been previously described.20 Briefly, fibrinogen adhesive was prepared from each patient by obtaining 45 ml of blood into a syringe containing 5 ml of 3.8% w/v sodium citrate. The blood was centrifuged, and the plasma was separated, frozen to −20°C then was allowed to thaw to 4°C overnight. The precipitated fibrinogen was separated out by further centrifugation at 4°C, and the supernatant serum was discarded. Then the cryoprecipitate was refrozen to −20°C and stored for up to 48 h.
When we mixed the fibrinogen (prepared from autologous blood by cryoprecipitate technique) with the commercially available thrombin (Fibri-Prest, Diagnostica Stago, Inc., Asnieres-Sur-Seine, France, 800 NIH-U/ml) a fibrin clot developed with a sufficient adhesive strength. None of the glue group required suturing.
Thrombin concentration can be varied to regulate the speed of coagulation. For example, low thrombin concentration (4 NIH-U/ml) with slow clotting is used in skin grafting while higher thrombin concentration (500 NIH-U/ml) is beneficial where almost instantaneous clotting is desired. Syringe applicator is usually advocated for mixing. However, the sealant can be applied sequentially or premixed through needles, spraying heads, or catheters. The glue does not stick to intact corneal or conjunctival epithelium.10
All surgeries were performed under local anesthesia (subconjunctival injection of Lidocaine HCl 20 mg/ml (Obarcaine 2%-Oubari Pharma-Syria) in out-patient settings. No patient required additional local anesthesia. No post-operative analgesic was required.
Our conventional surgical excision was started by shaving the lesion from the corneal side, starting half a millimeter in the clear cornea and finishing by excising the base of the lesion including the entire diseased tissue (keratinized conjunctiva and abnormal fibrovascular tissue). In recurrent cases, we tended to identify the medial rectus prior to lesion excision. We paid particular attention to the excision of the underlying and surrounding Tenon's capsule. Minimal cauterization was applied to bleeding vessels. The next step was to prepare the conjunctival autograft with the following method: The bare sclera (conjunctival defect) was measured, and then a free conjunctival graft was harvested from the superonasal bulbar conjunctiva. When harvesting the conjunctival graft, we aimed for a thin conjunctival layer with as little Tenon's tissue as possible, and for a graft that included a narrow edge of limbal corneal cells. The free graft was transposed, keeping it in the same orientation in relation to the cornea. The conjunctival defect where the graft was harvested from was left without suturing. At this stage, the conjunctival autograft was either sutured or glued. For suturing the graft, 8/0 vicryl sutures were used (PGA FSSB-Chirurgische Nadeln GMBH-Germany), so that four corners of the graft were anchored to the episclera with single sutures in addition to one continuous conjunctival-conjunctival suture, and knots were buried whenever possible. In cases of autologous fibrin glue, the graft was placed with the epithelium side down on the cornea, one drop of the thrombin component (800 NIH-U/ml) was placed on the scleral bed, and one drop of the fibrinogen solution was placed on the graft, and the graft was quickly flipped over the sclera and smoothed out while the fibrinogen was activated by the thrombin forming the fibrin glue. After positioning the graft, we allowed about 30 s to smooth out the graft and depressed it gently to the scleral bed, to attach it firmly but not stiffly. Excess glue was removed, and the graft was trimmed if necessary. Patients were discharged on the same day with the double eye patch for 24 h. We did not use bandage contact lenses.
Post-operative management included topical Dexamethasone-Neomycin Sulphate-Polymyxin B Sulphate eye drops (Alphaxitrol, Aleppo Pharmaceutical Industries-Alpha, Syria) instilled 6 times daily for 1 week. Post-operative medications were started on the 1st post-operative day and tapered gradually over 4 weeks.
Surgical time was noted from the first incision to the removal of the lid speculum. Post-operative pain was graded according to the VAS. Patients were followed-up after 1, 4, and 8 weeks and every 4 months thereafter. Sutures were only removed if the patient complained of irritation. Outcome measures included-in addition to success of the surgical procedure - post-operative pain, surgical time and recurrence rate. Statistical analysis was performed using the Mini-Tab v11 software. The Mann-Whitney (two-tailed) test was used with a 0.05 level of significance. When mean values were calculated, results were presented as mean ± standard deviation.

BODY.RESULTS:
A total of 54 patients (90 eyes) underwent surgical excision of the nasal pterygium [Table 1]. Fifty-eight percent of patients were males and 42% were females. The mean age was 38 ± 11.2 years (range, 20 years to 65 years). There were 42 primary and 48 recurrent nasal pterygia that were excised in a traditional manner. All patients received a conjunctival autograft from the same eye. Autologous cryoprecipitate was applied to 47 eyes (glue group), and sutures (suture group) were used in 43 eyes (suture group) to attach the free conjunctival graft. Follow-up ranged from 6 months to 18 months (mean, 12 ± 4.3 months).
Table 1Demographic of the glue and suture groups that underwent pterygium excision
The VAS results were collected from all patients. Adjusted VAS values were analyzed and compared between groups for each time the VAS was measured. The medians of the VAS values were significantly lower when glue was used (P < 0.05) [Table 2, Figure 1].
Table 2VAS values with the differences between two groups that underwent pterygium excision
Figure 1Medians of the post-operative visual analogue scale values in glue group (single line) and sutures group (double line)
The median surgical time was 11 min (range 9-15) for the glue group and 21 min (range 12-28) for the suture group (P <0.05) [Table 1]. No significant intraoperative or post-operative complications were noted. None of the patients in the glue group showed any reaction to autologous cryoprecipitate. The recurrence rate was 12%, and all recurrences occurred in cases of previously recurrent pterygium, in the suture group only during the early post-operative period (1st 3 months post-operatively). Cosmetic results in all other cases were excellent.

BODY.DISCUSSION:
The most commonly used tissue adhesive glue in ophthalmology is Tisseel Duo Quick (Baxter, Vienna, Austria), which is a two-component tissue adhesive that mimics the natural fibrin formation, and it is prepared from banked human blood. Autologous cryoprecipitate may be superior to commercial products since it probably reduces the risk of transfusion transmitted diseases. It has two modes of action: Sealing the wound and acting as a hemostatic agent. The rationale of using fibrin glue is to maximize post-operative patient comfort, to reduce suture related complications and less importantly to shorten the surgical time. In our case series, problems associated with suture closures of wounds (e.g., cysts, granulomas, button hole, and milia) were not seen. We did not notice any obvious transplant edema that was described in the other studies in either group.1021
VAS is commonly used to analyze and quantify the severity of pain. It can be difficult to perform in some patients. Careful instructions should be given to patients. In our study, adjusted VAS values showed a statistically significant difference in pain between the two groups (P < 0.05). The glue group had significantly lower pain levels as shown in Figure 1. As expected, surgical time was shorter in the glue group, almost half the time required to perform pterygium excision with sutured conjunctival autograft, and this was reflected on the amount of post-operative inflammation and pain. We also found that the size of the lesion did not affect the time of surgery nor caused more post-operative pain.
One of the most significant findings of our study was the low recurrence rate in the glue group, and that was further enhanced by the fact that most of our patients were a high-risk group (exposure to ultraviolet light as is the case of farmers or those who work outdoors, effects of dry hot weather and pollution on the ocular surface, and the poor compliance with treatment among a large group of our patients).
Usually, recurrence in most cases is seen within the 1st 6 months.22 Pterygia with a high tendency to grow can recur remarkably quickly after the surgical excision. This was the case in our group where all recurrences occurred in the 1st 3 months after surgery.
Most of the publications have described the use of gluing technique in primary pterygium management. This was the 1st time that autologous cryoprecipitate was used to attach conjunctival grafts after excision of primary and recurrent pterygia. The recurrence rate remained remarkably low at 12%, compared to 2-39% in other studies.21 It is postulated that gluing the conjunctiva inhibits fibroblasts migration into the surgical area, thus, minimizes the risk of recurrence.
What is new is that this technique was used for both primary and recurrent cases, by excising the pterygium and attaching the autograft using autologous cryoprecipitate tissue glue. All recurrences required further surgical excision, and we electively chose to use natural tissue glue in their management to further reduce the risk of recurrence.
If we consider the cost of the procedure, the costs of natural fibrin glue preparation are limited to in-house preparation of the glue, and then we can minimize the costs by preparing tissue glue for at least 4 patients at each time. Although in theory, this may increase the risk of a patient inadvertently receiving someone else's plasma, a strict protocol was used to avoid this problem. If we add to that the shorter surgical time, then it will be cost-effective to use natural tissue glue rather than suturing conjunctival grafts. Finally, this study meets the published report's recommendations that research should be dedicated to making fibrin glue available and affordable to more patients, and answers some of the concerns regarding the possibility of transmitting infectious agents through commercially available fibrin glue.23

BODY.CONCLUSIONS:
The use of autologous cryoprecipitate to attach the free conjunctival autograft in pterygium surgery appeared to be safe, reliable, quick, and repeatable with one of the lowest recurrence rates for both primary and recurrent pterygium surgeries. It produced less post-operative pain, and it required shorter surgical time, compared with conventional pterygium surgery. Moreover, there were no cases of recurrence during the follow-up period.

Question: Compared to absorbable sutures what was the result of autologous cryoprecipitate on The medians of the VAS values?
Evidence: The medians of the VAS values were significantly lower when glue was used (P < 0.05) [Table 2, Figure 1].
Answer: significantly decreased