Autologous in vitreo generated plasmin assisted vitrectomy in the pediatric population

Abstract

BACKGROUND

Inducing posterior vitreous detachment (PVD) is a crucial step in vitreoretinal surgery. However, compared with older patients, induction of PVD in pediatric and young adult populations is considerably more difficult. Recent studies have shown the utility of autologous plasmin enzyme in inducing PVD in pediatric patients.

AIM

To evaluate the anatomical and functional outcomes of patients with retinal detachment, treated with in vivo generated autologous plasmin, as an adjunct to vitreoretinal surgery.

METHODS

This is a retrospective observational case series including ten eyes of nine patients with retinal detachment, secondary to various pathologies. All eyes that received simultaneous intravitreal injection of 0.1 mL of autologous blood and 25 μg of tissue plasminogen activator (t-PA) 3 days before surgery were analyzed.

RESULTS

The mean age of the cohort was 9.3 +/- 4.16 years, ranging from 5 years to 16 years. Four out of nine patients were female. Five of the ten eyes had previously undergone vitreoretinal surgery. In the study cohort, five patients had high myopia, two patients had Stickler’s syndrome, one had juvenile X-lined retinoschisis, and one had familial exudative vitreoretinopathy associated with retinal detachment. No eye required repeat vitreoretinal surgery for recurrent retinal detachment or membrane peeling following the primary procedure, with adjunctive intravitreal autologous plasmin the median baseline visual acuity was 2.30 logarithm of the minimum angle of resolution (LogMAR) [interquartile range (IQR): 1.85-2.60], and the median final visual acuity was 1.54 LogMAR (IQR: 1.08-1.78), showing a statistically significant improvement (P = 0.01), with five eyes regaining functional visual acuity.

CONCLUSION

Adjunctive intravitreal autologous plasmin improves the anatomical outcome of vitreoretinal surgery in pediatric patients. However these findings require validation in a large sample, prospective study.

Key Words: Autologous whole blood; Enzymatic vitreolysis; Pediatric vitreoretinal surgeries; Plasmin; Retinal detachment; Tissue plasminogen activator

Core Tip: Posterior vitreous detachment (PVD), a key to the success in all vitreoretinal surgeries, is a crucial and challenging step in pediatric vitreoretinal surgeries especially syndromic retinal detachments. Surgical success is not fruitful even in experienced hands. The use of enzymatic vitreolysis (with plasmin) is promising in PVD induction, but unfortunately its usage is restricted due to the limitation in procuring these less durable enzymes. This method of in vivo plasmin generation by injecting a readily available agent (autologous whole blood) and tissue plasminogen activator yields plasmin inside the vitreous cavity and help in PVD induction henceforth surgical success. This retrospective pilot study will be beneficial for practicing vitreoretinal surgeons.

INTRODUCTION

Inducing posterior vitreous detachment (PVD) is a crucial step in vitreoretinal surgery. However, compared with older patients, inducing PVD in pediatric and young adult populations is considerably more difficult[1]. This is mainly attributed to the stronger vitreoretinal adhesion present in younger individuals. Even when a Weiss ring is produced through mechanical manipulation, the separation may be incomplete, representing only a lamellar split with residual cortical vitreous remaining adherent to the internal limiting membrane (ILM)[1].

Autologous plasmin has been widely used to facilitate the separation of cortical vitreous from the ILM. Previous studies have demonstrated its effectiveness in achieving complete PVD, leading to improved anatomical and visual outcomes in several pediatric vitreoretinal conditions, including macular holes[2,3], macular pucker (epiretinal membrane with ILM wrinkling or creases), persistent fetal vasculature, and rhegmatogenous retinal detachment (RRD)[4]. Its use has also been reported in the management of stage V retinopathy of prematurity, although the benefits appear limited[5]. Furthermore, autologous plasmin has shown utility in the treatment of proliferative membranes associated with proliferative diabetic retinopathy[6].

Despite these advantages, the clinical use of autologous plasmin is restricted by the complicated process required for its preparation and purification from blood samples[7,8]. In addition, the enzyme is unstable at room temperature and must be administered within a short period after preparation[3,4].

To address these limitations, the pilot study by Aras et al[9] reported that intravitreal injection of whole blood combined with tissue plasminogen activator (t-PA) can generate plasmin in vivo in pediatric patients.

Therefore, the present study aims to evaluate the efficacy of autologous plasmin injected into the vitreous cavity three days before surgery, to facilitate PVD induction in pediatric patients (< 16 years of age) with failed retinal detachment surgeries or in detachments primarily associated with syndromes such as familial exudative vitreoretinopathy (FEVR), Stickler syndrome and Juvenile retinoschisis.

MATERIALS AND METHODS

This is a retrospective observational case series conducted at the Aravind Eye Hospital, Coimbatore. The study was approved by the institutional Medical Ethics Committee and adhered to the principles of the World Medical Association Declaration of Helsinki. Written informed consent was obtained from the parents or legal guardians of all participants before the procedure.

The electronic medical records (EMR) of nine young patients comprising 10 eyes without pre-existing PVD who received an intravitreal injection of 25 μg t-PA with autologous whole blood about 2.5 mm to 4 mm from the limbus based on the age under short general anesthesia three days before vitrectomy were analyzed. These procedures were performed between December 2022 and June 2025 for various vitreoretinal pathologies. The study included both patients undergoing primary vitreoretinal surgery and those with a history of previous failed vitreoretinal surgery. Eyes with complicated cataracts were also included in the analysis. Patients older than 16 years and those with retinal detachment with extensive proliferative vitreoretinopathy (PVR) were excluded.

All patients underwent a comprehensive baseline ophthalmic examination, including visual acuity assessment using a Snellen chart, slit-lamp biomicroscopy, intraocular pressure measurement, and indirect ophthalmoscopy. Data collected from the EMR included age, sex, baseline and final visual acuity, underlying vitreoretinal pathology, history and number of previous vitreoretinal surgeries, and final anatomical outcomes. Visual acuity values were converted to logarithm of the minimum angle of resolution (LogMAR) units for analysis.

Functional low vision was defined as the ability to utilize residual vision for performing daily activities, particularly navigation. This functional measure emphasizes real-world visual performance rather than visual acuity alone. Patients with visual acuity ranging from < 6/18 to perception of light (PL⁺) were included, provided they demonstrated adequate performance in activities of daily living, in accordance with the World Health Organization consultation definition.

Five days prior to the intravitreal injection, patients were screened for seropositivity and systemic infections using blood and urine investigations. Then under strict aseptic precautions, 0.1 mL of autologous whole blood was obtained from the brachial vein and injected into the vitreous cavity about 2.5 mm to 4 mm from the limbus based on the age using a 27-gauge needle. Subsequently, 25 μg of (t-PA; Actylise, Boehringer Ingelheim, Ingelheim am Rhein, Germany) diluted in 0.05 mL of normal saline was injected intravitreally using a 30-gauge needle for in vivo plasmin generation. Anterior chamber paracentesis was performed if the intraocular pressure was digitally elevated. Topical antibiotics were prescribed for three days following the injection.

Three days after the injection, vitreoretinal surgery was performed under general anesthesia using the Constellation Vision System (Alcon, Fort Worth, TX, United States) with a 25-gauge vitrectomy system by a single experienced surgeon. PVD induction was performed using the vitrectomy cutter at a vacuum of 500 mmHg. Successful PVD was defined by the presence of a freely mobile Weiss ring separated from the retina. Staining with intravitreal diluted triamcinolone acetonide will help in the appreciation of the Weiss ring, and in the setting of the availability of intraoperative optical coherence tomography (OCT), more objective intraoperative validation is possible. However, intraoperative OCT was not used in the present study. Depending on the case requirements, the procedure was combined with pars plana lensectomy or scleral buckle surgery. At the end of surgery, long-acting tamponade with silicone oil (1500 centistokes (cs) or 5000 cs) was injected into the vitreous cavity.

Statistical analysis

The statistical analysis was performed using SPSS (SPSS v20.0, IBM Corp., Armonk, NY, United States). Snellen visual acuity was converted into LogMAR units. Normality of the data set was analyzed using the Shapiro-Wilk test. The Wilcoxon signed-rank test was used to compare preoperative visual acuity with postoperative visual acuity. A P value of less than 0.05 was considered statistically significant.

RESULTS

The study included 10 eyes in 9 patients, including five males and four females. One patient underwent bilateral sequential surgery. The mean age of the cohort was 9 ± 4.30 years (range: 5-16 years). Patient characteristics are summarized in Table 1.

Table 1 Patient characteristics.

Age	Gender	Eye	Diagnosis	Additional findings	Baseline visual acuity	Prior surgery	No: Of prior VR surgeries	Type of surgery	Additional surgery	Final visual acuity	Final anatomical outcome	Final functional outcome
5	Female	OD	Total RRD	Stickler syndrome	PL+	SB + PPV + SOI	2	SOR + MP + SOI	Cataract Sx + SOR	6/9	Attached	Yes
16	Male	OS	Total RRD	High myopia	3/60	Nil	Nil	PPL + PPV + SOI	Secondary IOL + SOR	6/18	Attached	Yes
5	Male	OS	Total RRD	JXLR	1/60	Nil	Nil	PPV + SOI	Cataract Sx + SOR	1/60	Attached	No
5	Male	OD	Total RRD	Stickler syndrome	PL+	PPV + SOI	2	SOR + MP + PFCL + SOI	PFCL removal + SOI	PL+	Attached	No
12	Male	OS	Total RRD	High myopia	HM	PPV + SOI	2	SOR + MP + SOI	Membranectomy	3/60	Attached	Yes
12	Female	OD	Complicated cataract + total RRD	Microcornea; high myopia	HM	PPL + PPV + SOI	1	SOR + MP + PFCL+ SOI	PFCL removal + SOI	PL+	Attached	No
		OS	Total RRD	Microcornea; high myopia	HM	Nil	Nil	PPV + SOI	PFCL removal + SOI	5/60	Attached	Yes
13	Male	OS	Total RRD	FEVR	3/60	SB	Nil	PPV + SOI	Partial SOR	5/60	Attached	Yes
8	Female	OS	Total RRD	High myopia	PL+	Nil	Nil	PPV + SOI		1/60	Attached	No
5	Female	OS	Total RRD	Ectopia lentis	HM	PPL + PPV + SOI	2	SOR + MP + SOI	SOR	1/60	Attached	No

OD: Oculus dexter; OS: Oculus sinister; RRD: Rhegmatogenous retinal detachment; JXLR: Juvenile X-linked retinoschisis; FEVR: Familial exudative vitreoretinopathy; PL: Perception of light; HM: Hand movements; SB: Scleral buckling; PPV: Pars plana vitrectomy; PPL: Pars plana lensectomy; SOI: Silicone oil injection; MP: Membrane peeling; PFCL: Perfluorocarbon liquid; SOR: Silicone oil removal; IOL: Intra ocular lens; Nil: Nothing; Sx: Surgery.

All patients underwent surgery for RRD. The underlying etiology of RRD were high myopia in five patients, Stickler syndrome in two (Figure 1A), and one case each of X-linked juvenile retinoschisis and FEVR. Five of the ten eyes underwent vitreoretinal surgery for the first time. One eye had undergone one previous vitreoretinal surgery, while the remaining four eyes had a history of two prior failed vitreoretinal surgical interventions.

Figure 1 Color fundus photograph of a Stickler syndrome patient with rhegmatogenous retinal detachment A: Pre-operative; B: Post-operative.

During surgery, three of the ten eyes required a pars plana lensectomy as they were associated with complicated cataracts. In all cases, PVD or PVR membrane removal was successfully achieved by the surgeon. At the conclusion of surgery, all eyes received postoperative silicone oil tamponade.

Cases were followed up for six months postoperatively. The mean baseline visual acuity was 2.30 LogMAR [interquartile range (IQR): 1.85-2.60], which improved to 1.54 LogMAR (IQR: 1.08-1.78), demonstrating a statistically significant improvement (P = 0.01). Anatomically, all eyes had an attached retina at the six-month follow-up. No patient required repeat surgery for recurrent retinal detachment after treatment with in vivo generated plasmin (Figure 1B).

No complications such as endophthalmitis, ghost cell glaucoma, or significant intraocular inflammation were observed following the intravitreal injection of autologous whole blood and t-PA.

DISCUSSION

In this study, we evaluated the effect of in vivo generated autologous plasmin in inducing PVD and its role in facilitating the separation of PVR membranes. The outcomes demonstrated anatomical success in 100% of operated eyes, with 50% of eyes achieving functional vision. The visual acuity on Snellen's chart ranged from > 3/60 to 6/9 (LogMAR: 1.30-0.2).

As induction of PVD is challenging in young patients due to strong vitreoretinal adhesions, many vitreoretinal surgeons prefer primary scleral buckling surgery as the first choice[10,11]. However, pars plana vitrectomy (PPV) is required in cases with posteriorly located retinal breaks or when scleral buckling surgery fails.

Among several enzymatic agents investigated to assist PVD induction, plasmin has shown the most promise. Plasmin is a non-specific serine protease that causes a dose-dependent and complete PVD[9]. It acts by degrading glycoproteins such as laminin and fibronectin, which act as primary adhesive components between the vitreous cortex and the ILM of the retina. This occurs either directly or through activation of endogenous matrix metalloproteinase-2[12,13].

Ultrastructural examination of post-mortem human eyes has demonstrated only sparse collagen fibrils on the ILM following intravitreal injection of plasmin[14]. However, no vitreous detachment was observed at the vitreous base[14]. Additionally, plasmin does not degrade type IV collagen, which is a major structural component of the ILM, thereby preserving a smooth retinal surface[15].

Despite these advantages, the clinical use of plasmin is limited by the complex process required for its purification from blood and its relative instability at room temperature[4]. Chuang and Chen[16] demonstrated that intravitreal injection of t-PA in eyes with vitreous hemorrhage could induce PVD in pediatric patients undergoing vitreoretinal surgery. The authors proposed that t-PA facilitates the conversion of plasminogen present in intravitreal blood into plasmin within the vitreous cavity.

Based on these findings, Aras et al[9] induced pharmacological vitreolysis by injecting autologous whole blood, a source of plasminogen, along with t-PA into the vitreous cavity of young patients scheduled for vitrectomy for various indications.

In our study, 0.1 mL of autologous whole blood and 25 μg of t-PA were injected into the vitreous cavity three days prior to the scheduled vitreoretinal surgery. Injecting blood into the vitreous cavity can lead to short-term complications such as rapid clot formation, slow lysis of fibrin, extracellular lysis of red blood cells, persistence of intact red blood cells for months and lack of early polymorphonuclear response. Long-term complications include hemosiderosis bulbi, retinal damage, glial and fibrovascular proliferation and glaucoma (ghost cell/hemolytic/hemosiderotic)[17,18]. Usage of a minimal amount of blood (0.1 mL), along with t-PA, addresses the unique catabolic features of blood in the vitreous cavity such as rapid clot formation, slow lysis of fibrin and extracellular lysis of red blood cells. As surgery was performed within 3 days, there is no issue of long-term complications like ochre membrane, PVR or glaucoma. Furthermore, following intravitreal injection of t-PA, maximum plasmin generation from plasminogen occurs within approximately 2-5 hours[19]. By delaying surgery for three days, it is presumed that plasmin binds more extensively to substrates at the vitreoretinal interface, thereby enhancing its efficacy[9].

t-PA (Actylise, Boehringer Ingelheim, Ingelheim am Rhein, Germany) is supplied as a 20 mg vial costing approximately $230 in India. After reconstitution, the drug loses its potency within 24 hours. For intravitreal plasmin generation, only 25 μg is required per dose, and the remaining drug must be discarded as it cannot be reused. To reduce the cost burden, we aliquot the 20 mg vial into 1 mg portions under laminar airflow and store them as cryoprecipitates, allowing the injection to be offered at a reduced cost of approximately $32 per dose.

Ocriplasmin (microplasmin) is highly expensive, costing approximately $3950 per dose. The aliquots of Actilyse serve as a cost-effective alternative. To understand this better, a structured cost-comparison analysis of the three drugs is described. Procedure A: Actilyse whole vial (cost-$230); Procedure B: Aliquots of Actilyse (cost-$32); Procedure C: Ocriplasmin (cost-$3950).

Cost-comparison analysis shows that Procedure B is significantly cheaper than procedures A and C, with the difference entirely attributable to higher drug costs. These findings are particularly relevant in resource-constrained settings, where minimizing healthcare expenditure without compromising outcomes is critical.

Compared with autologous serum or plasma, autologous whole blood contains higher concentrations of plasminogen and fibrinogen, which facilitate the activation of t-PA[9,20].

Notably, complete PVD was achieved in all cases undergoing primary vitrectomy. This finding is encouraging as PVD induction is one of the most critical steps in vitrectomy, and incomplete removal of cortical vitreous is a major factor contributing to surgical failure.

Although PVR is typically considered a contraindication for in vivo plasmin generation due to the presence of t-PA inhibitors that limit the conversion of plasminogen to plasmin and thereby reduce the efficacy of enzymatic vitreolysis[9], our observations suggest that this approach may still be beneficial in cases of failed vitreoretinal surgery associated with incomplete vitreous separation and early PVR formation. Previous studies have evaluated the effect of intravitreal plasmin in the management of proliferative membranes in proliferative diabetic retinopathy[6,21].

Autologous plasmin-assisted vitrectomy has been reported for macular holes, macular pucker, persistent fetal vasculature, RRD, stage 5 ROP, and proliferative membranes in diabetic retinopathy[2-6,20] A detailed literature search did not show the usage of enzymatic vitreolysis by in vivo plasmin generation in failed retinal detachment surgeries. To the best of our knowledge, not many studies have evaluated this approach in the context of failed vitrectomy surgeries.

Although anatomical success was achieved in 100% of cases, only 50% of eyes achieved functional vision. The main reason for this was chronicity (50% failed primary surgery), severity of the condition and amblyopia (as pediatric cases with unilateral pathology present late).

Limitations: This study is subject to several important limitations. First, its retrospective design inherently restricts the ability to infer causal relationships. Second, the absence of a control group precludes direct comparison with standard PPV alone. Furthermore, the relatively small sample size limits the external validity and generalizability of the findings. The inclusion of patients with diverse underlying pathologies may have introduced confounding variables that could have influenced the observed outcomes. In light of these limitations, the results should be interpreted with caution, as they appear to be acceptable but not demonstrably superior to the expected baseline outcomes.

Our method shows that the need for a cumbersome process to extract plasmin can be avoided and also serves as a cost-effective alternative to Ocriplasmin. However, a larger, structured, prospective study including a comparison with Ocriplasmin will be more beneficial to prove the efficacy of in vivo plasmin.

CONCLUSION

In vivo generated autologous plasmin, from intravitreally injected autologous whole blood and t-PA, is a cost-effective adjuvant to achieve complete PVD in the pediatric population. However these results need to be validated in a controlled study with a larger sample size.