Best Autologous Serum Preparation Protocol

Autologous serum helps patients with many ocular inflammatory diseases, such as Stevens-Johnson Syndrome, Graft Versus Host Disease, Chemical burns, dry eye, and eye infections. I have used it for years since my days at Harvard. At Visionary, our team has used it for over 20 years with excellent results.

It does not work on 100% of patients but over 90% of patients find relief with their own serum alone. Some patients need to use their own platelets as Platelet Rich Plasma before they find relief of eye symptoms. But interestingly enough, they get relief when they go back to their own serum where it did not work before. 

What is the best way to prepare Autologous Serum:
This is a controversial question. There are many protocols on how to prepare serum: how long to clot; how long to use before throwing out the bottle; how many times to use per day; what percent serum should be used. 

Below are many references to show many various techniques. 

Both at Harvard and at Visionary, we have used 1 bottle of serum for 5-7 days while being kept defrosted in the refrigerator (at all times unless putting it in the eye) for years. 

Also both at Harvard and at Visionary, the clotting usually takes 5-10 minutes. I could not find a benefit of waiting 6-8 hours to clot like some protocols use below. Blood clots in a short amount of time unless there is a clotting disorder, in which case, we would wait longer but have not seen this yet. 

Generally, most studies note that 20% works well. We have noted a few patients (less than 5% of our patients) who only get relief at 50% or 75% or 100%. I do not know why this happens to some patients. We suspect smokers or patients on a high inflammatory diet (ie, eats a good deal of carbohydrates, sugar, gluten), may be less likely to benefit from their own serum, but I have no proof on this yet. 

Also, generally we start 20% autologous serum 6-8 times per day until pain/discomfort scores decrease to about a 2-3 out of 10. Once this occurs, patients can start to decrease to:
6x/day for 1 week, 
then 5x/day for 1 week,
then 4x/day for 1 week, and then continue to taper down, while maintaining their use of nonpreserved artificial tears and /or Xiidra if using to find a “sweet spot” of where they are having minimal to no ocular discomfort with the least amount of serum or prescription drops. 

There are risks with using your own serum, namely a severe corneal infection. It has been reported. I have never seen one not have most of my colleagues. Thus some pharmacies recommend discarding each bottle after 3 days. Or some recommend using an antibiotic drop every day once a day to protect against infection. 

There is no standardized way to make or use Autuologous Serum. Even meta-analysis papers lament this. 
Thus often a patient has to try to see how his or her eye will react. 

Autologous serum is generally safe. I would not recommend using someone else’s serum for now.


Example: References:

Method of preparation for autologous serum eye drops. Autologous serum eye drops were prepared using autoserum collected from each patient in sterile conditions.

Table I

Characteristics and main results of the included randomised controlled trials (RCTs) on the use of serum eye drops in ocular surface diseases.
Study (year)ref Patients (n) Median age, years (range) Condition Product Concentration (frequency) Control arm Duration of treatment/follow up Concomitant topical therapy Main results
Tananuvat (2001)15 12 59.5 (33–80) SS, NHL, GvHD, SJS, RA AS 20% (6 times/day) NSS 2 months None AS group had no statistically significant improvement in symptoms and objective signs (IC, FS, RBS, ST, TBUT) of dry eye
Vajpayee (2003)16 59 47.8 (19.8)1 CED CBS 20% (6 times/day) AS 3 weeks None Higher % of re-epithelisation in the CBS group
Noble (2004)17 16 54 (30–71) GvHD, SS, OCP, AS 50% (NA) CT 3 months None Significant improvement in symptoms and IC
Kojima (2005)18 37 NA SS, non-SS AS 20% (6 times/day) AT 2 weeks None Significant improvement in symptoms and TBUT, RBS, FS
Schulze (2006)19 23 64.8 (9.6)1 DCL AS 100% (hourly) Hyaluronic acid drops Variable (until healing) Isoptomax, atropine, neosynephrine AS led to a significantly quicker closure of corneal epithelial wounds
Noda-Tsuruya (2006)20 27 30.1 (5.8)1 Post-LASIK dry eye AS 20% (5 times/day) AT 6 months None Significant improvement in TBUT, FS and RBS. No change of symptoms
Sharma (2011)21 32 NA OCI CBS 20% (10 times/day) AS, AT 3 months Ofloxacin, prednisolone, homatropine hydrobromide, sodium citrate Significantly higher % of corneal transparency in CBS group
Urzua (2012)22 12 52 (6.3)1 DES AS 20% (4 times/day) AT 2 weeks None Significant improvement in subjective (OSDI), but not objective (FS and TBUT) scores
Panda (2012)23 20 NA OCI PRP – (10 times/day) CT 3 months None Significantly faster epithelial healing and improvement in cornea transparency in PRP group
Cho (2013)24 85 NA SS, non-SS, PED AS 100% (6 times/day) AS (NSS, hyaluronic acid, ceftazidime) 3 months None In SS patients, undiluted AS was the most effective in decreasing symptoms, corneal epitheliopathy and promoting fast closure of wound
Lopez-Garcia (2014)25 26 52 (13.4)1 SS AS (sodium hyaluronate) 20% (3 times/day) AS (NSS) 2 months None Significant improvement in subjective symptoms and objective parameters (FS, RBS, TBUT, ST) in group with AS diluted with sodium hyaluronate
Celebi (2014)26 20 56 (8.0)1 DES AS 20% (4 times/day) AT 1 month None Significant improvement in OSDI and TBUT scores in AS group
Mukhopadhyay (2015)27 144 NA HD CBS/AS 20% (6 times/day) AT 6 weeks None CBS/AS therapy improved clinical parameters and tear protein profile in comparison with AT
Li (2016)28 37 48.3 (28–62) SS AS 50% (8 times/day) BCL 6 weeks Fluorometholone Patients in the BCL group had better OSDI and FS scores than patients in AS group
Lee (2016)29 21 NA PED AS+SCL 20% (12 times/day) No treatment after healing 3 months Levofloxacin Prolonged use of AS-SCL decreased recurrence rates
Yilmaz (2016)30 24 25 (4)1 DES AS 40% AT 2 months None Significant improvement in OSDI and TBUT scores in AS group
Semeraro (2016)31 24 NA SS AS – (5 times/day) AT 1 year None Significant improvement in OSDI in AS group
Sul (2018)32 50 NA PS AS 50% (8 times/day) AT 6 months Dexamethasone, antibiotics AS accelerated corneal epithelial healing with reduced pain following PS
Akcam (2018)33 60 NA PRK AS 20% 12 times/day) AT 1 year Moxifloxacin, dexamethasone AS accelerated corneal epithelial healing with reduced pain following PRK

AS: autologous serum; AT: artificial tears; BCL: bandage contact lens; BS: Behcet’s syndrome; CBS: cord blood serum; CED: corneal epithelial defects; CT: conventional treatment; DCL: diabetic corneal lesions; DES: dry eye syndrome; FS: fluorescein staining; IC: GvHD: Graft-versus-Host Disease; HD: Hansen’s disease; impression cytology; NA: not available; NHL: non-Hodgkin lymphoma; NSS: normal saline solution; OCI: ocular chemical injury; OCP: ocular cicatricial pemphigoid; OSDI: ocular surface disease index; PED: persistent epithelial defect; PRK: photorefractive keratectomy; PRP: platelet-rich plasma; PS: pterygium surgery; RDS: Riley-Day syndrome; RBS: rose Bengal staining; SCL: soft contact lenses; SJS: Steven-Johnson syndrome; SS: Sjogren’s syndrome; ST: Shirmer test; TBUT: tear break-up time. 1Mean age (standard deviation).

Special Issue  |   November 2018

Autologous Serum and Serum Components
 Author Affiliations & Notes
Investigative Ophthalmology & Visual Science November 2018, Vol.59, DES121-DES129. doi:10.1167/iovs.17-23760

Dry eye syndrome is a multifactorial condition on the tear and ocular surface. Autologous serum eye drop is an effective method for treating dry eye. Autologous serum eye drops are now widely used by specialists since a first report in 1975. The results of a systematic study showed that the efficacy of autologous serum eye drops remains ambiguous because its preparation methods and clinical application have not been standardized. To elucidate the efficacy of autologous serum eye drops, well-designed, large-scale, high-quality randomized controlled trials need to be conducted with standardized treatment and use. Since serum components are partially similar to tear components, autologous serum eye drops improve dry eye by supplying tear components such as growth factors, proteins, and vitamins. Adding to the evidence based on the treatment of dry eye, we have found a new treatment candidate from serum: selenoprotein P (SeP). The efficacy of SeP as a treatment for dry eye was revealed by applying SeP eye drops to a dry eye rat model. Compared with phosphate-buffered saline treatment, SeP eye drops significantly reduced the fluorescein score of the cornea and suppressed the oxidative stress in the cornea, which is related to onset of dry eye, leading to improved corneal disorder. We have developed a new dry eye model caused by oxidative stress that will be used to screen candidate molecules for antioxidative activity.
 1999 Apr;83(4):390-5.

Treatment of dry eye by autologous serum application in Sjögren’s syndrome.



To evaluate the efficacy of autologous serum application for the treatment of dry eye in Sjögren’s syndrome.


The stability of essential components (EGF, vitamin A, and TGF-beta) in preserved serum were examined following preservation at 4 degrees C and -20 degrees C. In a primary clinical trial, 12 patients with Sjögren’s syndrome were treated with autologous serum (diluted to 20% with sterile saline) for 4 weeks, and vital staining of the ocular surface was compared before and after treatment. The effects of serum on mucin (MUC-1) expression were observed in cultured conjunctival epithelial cells in vitro.


EGF, vitamin A, and TGF-beta were well preserved for up to 1 month in the refrigerator at 4 degrees C and up to 3 months in the freezer at -20 degrees C. Rose bengal and fluorescein scores improved significantly from the initial scores of 5.3 and 5.6 to 1.7 and 2.5 after 4 weeks, respectively. The additive effect of human serum for cultured conjunctival epithelial cells showed significant MUC-1 upregulation on the cell surface.


Autologous serum application is a safe and efficient way to provide essential components to the ocular surface in the treatment of dry eye associated with Sjögren’s syndrome.

Materials and methods
Tears contain essential components for the
ocular surface such as EGF, vitamin A,
TGF-â, fibronectin, and various other cytokines. Since these components are also found
in serum, we formulated artificial tears by
diluting serum obtained from SS patients after
informed consent was obtained. A total of 40
ml of blood was procured by venepuncture and
centrifuged for 5 minutes at 1500 rpm. The
serum was carefully separated in a sterile manner and diluted by saline to 20%. The final
preparation was aliquoted into 5 ml bottles
with ultraviolet light protection since vitamin A
is easily degraded by light. Patients were
instructed to keep the bottle in a dark and cool
place, such as a refrigerator, while in use with
the rest being stored in a freezer until required.
Serum drops were applied 6–10 times a day in
addition to the old regimen of frequent
preservative-free artificial tears, highly viscous
hyaluronic acid (Hyalein, Santen Pharmaceutical Co, Osaka, Japan) four times daily, and the
use of special dry eye glasses for added humidity. These glasses maintained the moisture level
around the eye at 40–80%, depending on the
ambient humidity, while the 0.3% hyaluronic
acid oVered additional lubrication.17 Patients
were instructed to use autologous serum
(diluted to 20% with saline), 6–10 times a day
for 4 weeks and vital staining of the ocular surface was compared before and after treatment.

. 2016; 9: 47–54.
Published online 2016 Mar 2. doi: 10.2147/IMCRJ.S97297
PMCID: PMC4780666
PMID: 27042143

The use of autologous serum for the treatment of ocular surface disease at a Swedish tertiary referral center

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Treatment protocol

AS drops were prepared by taking 10 mL of peripheral blood from each patient. The samples were allowed to clot for 30 minutes and were then centrifuged for 9 minutes at 2,900× g. Serum was extracted under sterile conditions. The serum (2 mL) was diluted with 8 mL of saline solution for a final concentration of 20%. One patient who did not respond to 20% serum was also treated with 100% serum. No testing for blood-borne diseases was performed. Antibiotics were not added to the solution but were included in the treatment regimen in 24 of 32 eyes. The bottles were frozen at −20°C and patients were instructed to store bottles in the freezer, keep current bottle in the refrigerator, and discard it after 1 day. Dosage of drops varied between patients from two to eight times daily, as shown in Table 1.

3. Autologous serum eye drop processing and testing


Serum eye drop preparation in Australia: Current manufacturing practice


Serum eye drops are used to treat diseases such as dry eye syndrome (keratoconjunctivitis sicca), a disease of the surface of the eye that results in an unstable tear film. Patients are referred to the Australian Red Cross Blood Service by ophthalmologists for autologous serum eye drops when other therapies such as artificial tears or topical immunosuppressive agents have failed. In order to manufacture autologous serum eye drops, whole blood is collected from the patients using standard blood collection procedures. The blood is then allowed to clot to produce serum and processed into 20% serum eye drops, which are then returned to the patient for their own use. The eye drops are packaged into a long length of tubing, which is then heat-sealed to produce single-use segments. The demand for serum eye drops in Australia is increasing every year, with a 30% increase in the past 12 months.
Serum is prepared from whole blood collected into a triple dry pack without anticoagulant. The blood is held for a minimum of 6 hours at room temperature to allow clot formation and retraction to occur, after which the blood is centrifuged to obtain serum and then separated and diluted 1:4 with 0.9% saline to produce 20% serum. The eye drops are packaged into a 20 metre length of tubing, which is heat-sealed to produce single-use, single-dose tubing segments (approximately 7 cm each) and stored below −18 °C prior to transport to the hospital nearest the patient for collection.
A standard blood collection provides approximately 1200 segments, which, depending on the frequency of application, can provide the patient with up to 1 year’s supply of eye drops. These segments are stored at or below −18 °C (the temperature of a domestic freezer), and have a shelf-life of 365 days post-collection if stored at this temperature. Each day, enough segments for 24 hours’ use can be removed from the freezer and placed in the patient’s refrigerator to thaw. As the serum does not contain any anti-microbial agents, unopened segments must be used within 24 hours of thawing, and opened segments must be discarded immediately after use. The dose of autologous serum eye drops to be administered and the frequency and duration of treatment depend on the clinical needs of each patient.

. 2004 Apr; 88(4): 464–465.
PMCID: PMC1772118
PMID: 15031155

A protocol for low contamination risk of autologous serum drops in the management of ocular surface disorders

Preparation of autologous serum drops

Autologous serum for use as eyedrops was prepared by obtaining approximately 30 ml of the patient’s blood in sterile vacutainer tubes without additive. The blood was left to clot at 4°C for 10 to 12 hours before centrifuging at 4500 rpm for fifteen minutes. The serum was separated in a laminar flow cabinet and diluted with sterile saline (0.9%) to a 20% concentration and aliquoted in 2.0 ml portions clearly labelled with the patient’s details. The vials were kept frozen at −20°C. A fresh bottle was thawed and used for treatment every 24 hours. All patients were on topical preservative free chloramphenicol drops.

 2016 Jul 4;7(7). pii: E113. doi: 10.3390/mi7070113.

Microfluidic Autologous Serum Eye-Drops Preparation as a Potential Dry Eye Treatment.

Yasui T1,2,3Morikawa J4Kaji N5,6Tokeshi M7,8Tsubota K9Baba Y10,11,12.


Dry eye is a problem in tearing quality and/or quantity and it afflicts millions of persons worldwide. An autologous serum eye-drop is a good candidate for dry eye treatment; however, the eye-drop preparation procedures take a long time and are relatively troublesome. Here we use spiral microchannels to demonstrate a strategy for the preparation of autologous serum eye-drops, which provide benefits for all dry eye patients; 100% and 90% removal efficiencies are achieved for 10 μm microbeads and whole human blood cells, respectively. Since our strategy allows researchers to integrate other functional microchannels into one device, such a microfluidic device will be able to offer a new one-step preparation system for autologous serum eye-drops.


autologous serum eye-drops; dry eye; spiral microchanne

Autologous serum eye-drops are a good candidate for dry eye treatment since they contain epidermal growth factor (EGF), vitamin A, and so on, which is essential for cell differentiation and division [,,]. Treatment using the autologous serum eye-drops is based on the concept that dry eye worsening is not due to drying out the front surface of the eye, but rather to poorly supplying essential components to the cornea; therefore, the autologous serum eye-drops can treat dry eyes comprehensively, by not only lubricating the front surface of the eye but also promoting cornea regrowth by the EGF []. Autologous serum eye-drops have two features. One is that users can reduce the chance of infection because the person’s own blood is utilized, and the other is that the eye-drops can be stored for up to three months at −80 °C. The autologous serum eye-drops are prepared as follows: first, a patient’s blood is collected in a heparin-unmodified blood collection tube; secondly, the collected blood is centrifuged at 3000 rpm for 10 min; thirdly, the supernatant is filtered through a 0.45-μm-pore-size filter; and finally, the filtered serum is diluted to reach a target concentration using saline. However, the preparation is relatively troublesome and takes a long time due to the centrifugation, filtration, and dilution steps.

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