Serum has several components that make it beneficial for ocular application in certain ocular disorders. This is because many patients with certain ocular disorders are lacking sufficient tear production and serum is similar to natural tears in many ways (Table 8.1). Serum has many components that enhance migration and proliferation of epithelial cells and have antimicrobial activity (Table 8.2). The most important of these factors are epithelial growth factor (EGF) and transforming growth factor beta (TGF-β). EGF accelerates the migration of epithelial cells, has anti-apoptotic effects, and is present in higher concentrations in serum compared to natural tears. Likewise, TGF-β is involved in the epithelial and stromal repair processes and its concentration in serum is three times higher than in natural tears. Other important components of serum that are also found in natural tears include vitamin A, albumin, α2-macroglobulin, fibronectin, substance P, insulin like growth factor 1, and immunoglobulins (Quinto et al. 2008).

EGF, epithelial growth factor; TGF-β, transforming growth factor beta.

EGF, epithelial growth factor; TGF-β, transforming growth factor beta; IGF-1, insulin-like growth factor 1; Ig, immunoglobulin.

Proteolytic enzymes, including tear film matrix metalloproteinases, are needed to maintain the slow turnover and remodeling of the corneal stroma and are balanced by natural proteinase (proteolytic) inhibitors in tears; these include plasmin, α1-proteinase inhibitor, α2-macroglobulin, and tissue inhibitors of metalloproteinases (Twining et al. 1994a,b; Hibbets et al. 1999). When there is an abundance of proteinases that exceeds proteinase inhibitors in the tears, rapid pathologic degradation of corneal stromal collagen and proteoglycans can occur, resulting in keratomalacia or “corneal melting” (Matsubara et al. 1991; Geerling et al. 1999). An imbalance characterized by high levels of proteinases in comparison to proteinase inhibitors is associated with the development of severe “melting” corneal ulcers (Figure 8.1) and certain types of superficial non-healing ulcers in horses and dogs (Ollivier et al. 2004; Bentley 2005; Wang et al. 2008).

Preparation procedure

Protocols for the preparation of autologous serum for ocular application vary significantly in the human literature (Table 8.3). The volume of blood collected from people ranges between 50–100 mL and an entire standard blood collection (400–450 mL). The volume of blood collected from animals depends on the size of the animal and the volume of autologous serum desired, although recommendations are lacking in the literature.

BES, balanced electrolyte solution.

A standard protocol for autologous serum preparation is not readily identifiable in the veterinary literature, but a protocol based on anecdotal experience and expert opinion is outlined in Box 8.1. In small veterinary patients, a blood collection for autologous serum preparation should not exceed 10% of blood volume, defined as 80–90 mL/kg in dogs and 45–60 mL/kg in cats. Ideally, patients should have undergone the same infectious disease screening that a blood donor would have undergone in order to ensure they are healthy enough for blood collection (see Chapters 13 and 15). Once the blood is collected sterilely into a syringe or container that does not contain anticoagulant, it should be allowed to clot prior to centrifugation. Once the serum is separated and removed, some recommend filtration to remove fibrin strands, which are suspected to reduce efficacy (Fox et al. 1984). Whether or not to dilute the serum in 0.9% saline, balanced electrolyte solutions, or antibiotic eye drop solutions is controversial and at the discretion of the clinician, as dilution with antibiotics might reduce the epitheliotrophic capacity of the serum, but the high viscosity of the concentration (non-diluted) solution can be irritating (Geerling et al. 2004; Quinto et al. 2008). Likewise, use of serum that is hemolyzed also causes ocular irritation and should be avoided (C.L. Pinard, personal communication 2015). Most veterinary ophthalmologists do not routinely filter or dilute the separated serum prior to administration (B.S. Bauer, personal communication 2015).

Storage and stability

Autologous serum, in its diluted or undiluted form, should be aliquoted into smaller volumes, which are stored at –20°C or colder until their use. Once thawed, the aliquots should be kept refrigerated (4°C) in between applications and kept away from light to avoid degradation of vitamin A (Quinto et al. 2008). Owners should be notified of the importance of storage temperatures to reduce bacterial growth if aliquots are stored at home rather than in laboratories or hospitals.

EGF, TGF-β, and vitamin A are stable in autologous human serum aliquots for 1 month under refrigeration and for 3 months if frozen (Tsubota et al. 1999a, 1999b). A study investigating the effect of storage temperature (–20°C vs –80°C) and duration of storage (0, 30, 90, and 180 days) using canine, feline, and equine autologous serum to prevent corneal damage found that storage time and temperature did not affect in vitro corneal weight loss and feline serum was the most protective (Conway et al. 2014). This suggests that autologous serum can be stored at temperatures of –20°C or colder for 6 months and maintain sufficient anti-proteinase activity.

Sterility

Autologous serum eye drops must be prepared using sterile technique. Due to concerns of bacterial contamination of dropper bottles during use, ideally an aliquot of prepared autologous serum is submitted for bacterial culture and the aliquots only used for ocular application once the bacterial culture is considered negative (Quinto et al. 2008). Recommendations regarding the duration of time an aliquot should be opened and used vary considerably from 16–24 hours to 1 week (Geerling et al. 2004). Ideally, a new aliquot in the form of an eye drop bottle or syringe should be used each day by owners, given that autologous serum eye drops used at home are more likely to have microbial contamination leading to microbial keratitis (Poon et al. 2001). Conversely, when used in hospital by trained personnel, bacterial contamination of eye dropper bottles does not typically occur until after day 4 (Sauer et al. 2004). To avoid the development of bacterial infections, it is recommended that patients receiving autologous serum eye drops also receive concurrent topical antibiotic therapy, if not otherwise indicated for their underlying ocular condition (Yamada et al. 2008).

Indications

Autologous serum eye drops are most commonly indicated in veterinary patients with ulcerative keratitis to reduce proteinase activity (Ollivier et al. 2007), particularly in animals with melting ulcers (Figure 8.2). Restoring the balance between anti-proteinase and proteinase activity helps to reduce proteolytic activity and subsequently improve epithelial healing and decrease corneal scarring (Berman 1975; Berman et al. 1975). Autologous serum is usually instilled into the affected eye every 1–2 hours until healing is appreciated by a reduction in ocular discomfort or improvement in the ulcer itself. Thereafter, application can be decreased to every 4–6 hours and usually does not exceed 72 hours of treatment (Ollivier et al. 2007). Assessment by a veterinary ophthalmologist is also recommended for patients with complex corneal ulcers, as surgical repair (e.g., conjunctival graft) is often required, especially in patients with significant stromal loss, which can result in corneal perforation and loss of vision or the eye within 24 hours (B.S. Bauer, personal communication 2015).

Autologous serum has also been compared to commercial ointment containing vitamin A, E, and hydrolyzed casein for the treatment of uncomplicated corneal ulceration in dogs. In addition to standard treatment, 41 dogs were randomized to receive two drops of autologous serum or 1 cc of the commercial ocular ointment every 6 hours and 10 minutes after antimicrobial drops. No difference in ulcer healing was noted between the groups of dogs; therefore, autologous serum was considered as effective as the commercial product for the adjunctive treatment of corneal ulceration in dogs (Ortiz et al. 2012).

Autologous serum eye drops have also been used in people with various causes of dry eye, persistent epithelial defects, graft-versus-host disease, neurotrophic keratitis, keratoconjunctivitis, recurrent corneal erosions, ocular surface reconstruction, aniridic keratopathy, and after laser in situ kertomileusis (LASIK) surgery (Quinto et al. 2008, Yamada et al. 2008). Given the conflicting evidence that autologous serum eye drops are superior to other forms of treatment, they are typically only recommended for these conditions when conventional forms of treatment have failed.

Contraindications

Autologous serum eye drops should not be prepared using patients with significant cardiovascular disease, anemia (packed cell volume (PCV) <30%), active bacterial, viral, or fungal infections, or whose infectious disease screen is not negative. Likewise, patients that have clinical conditions during which harmful substances might be present in the autologous serum should also be excluded. Examples include hyperbilirubinemia, hyperproteinemia, or patients receiving concurrent medications that might be irritating or toxic to the cornea (e.g., fluoroquinolones) (Yamada et al. 2008).

Complications

Complications are reported to occur in approximately 13% of human patients administered autologous serum eye drops of which 3% had an inflammatory response and 1.5% developed an infection. Other reported complications include scleral vasculitis, immune complex deposition, peripheral corneal infiltrate, corneal ulcer, and discomfort/irritation (Geerling et al. 2004; Yamada et al. 2008). Complications have not been reported for veterinary patients and are considered rare.

Cost

Cost analysis taking into account personnel and disposable materials used in the preparation of autologous serum eye drops demonstrates that the cost per 2 mL aliquot is approximately equivalent to one bottle of preserved pharmaceutical ocular lubricant (Geerling et al. 2004). However, depending on the frequency that autologous serum eye drops are prepared, as well as the comfort and skill level of the personnel involved, the time it takes might be sufficiently longer such that the cost is increased.

Conclusions

Despite relatively few veterinary publications documenting its clinical efficacy, autologous serum is used commonly in animals for the treatment of severe ulcerative keratitis. Approximately 80% of human patients with corneal epithelial disorders demonstrate some level of symptomatic relief in response to autologous serum eye drops reported in the small clinical trials and case series published (Yamada et al. 2008). However, some studies demonstrate a lack of effect based on objective clinical measures (Pan et al. 2013). Large, randomized, controlled clinical trials are needed in both human and veterinary medicine before autologous serum eye drops are used routinely, except in patients who have failed conventional forms of treatment.