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PERSPECTIVE
Autologous serum eye drops for ocular surface disorders
G Geerling, S MacLennan, D Hartwig.............................................................................................................................. .
Br J Ophthalmol 2004;88:14671474. doi: 10.1136/bjo.2004.044347
Tears have antimicrobial, nourishing, mechanical, and
opticalproperties. They contain components such as growth
factors, fibronectin, and vitamins to supportproliferation,
migration, and differentiation of the corneal and
conjunctival epithelium. A lack ofthese
epitheliotrophic factorsfor example,
in dry eye, can result in severe ocular
surface disorders such aspersistent
epithelial defects. Recently, the use of
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Geerling, MacLennan, Hartwig22
autologous serum in the form of eye drops has been
reported as a new treatment for severe ocular surface
disorders. Serum eye drops may beproduced as an
unpreserved blood preparation. They are by nature non-
allergenic and theirbiomechanical and biochemical
properties are similarto normal tears. In vitro cell culture
experiments showed that corneal epithelial cell morphologyand function are bettermaintainedby serum than by
pharmaceutical tearsubstitutes. Clinical cohort studies have
reported its successful use for severe dry eyes and persistent
epithelial defects. However, the protocols toprepare and
use autologous serum eye drops varied considerably
between the studies. As this can result in different
biochemicalproperties protocol variations may also
influence the epitheliotrophic effect of the product. Before
the definitive role of serum eye drops in the management of
severe ocular surface disease can be established in a large
randomised controlled trial this has to be evaluated in more
detail. In view of legislative restrictions and based upon theliterature reviewed here a preliminary standard operating
procedure for the manufacture of serum eye drops is
Nutrition of the ocular surface
While the corneal demand for glucose, electro-lytes, and amino acids is supplied by the aqueous
humour, growth factors, vitamins and neuropep-tides, which are secreted by the lacrimal gland,support proliferation, migration, and differentia-
tion of the ocular surface epithelia. 37 As part
of inflammatory processes
additional proteins such as theadhesion factor fibronectin,complement factors, and
antimicrobial proteins (forexample, lactoferrin,
immunoglobulins) are releasedinto the tears from conjunctivalvessels.8 9 Tears thus have
lubricating, mechanical, but also
epithelio- trophic and antimicrobial
properties. A reduction of
epitheliotrophic factors or their
carrier com- promises, as in the
gastrointestinal tract, the integrityof the surface epithelia. This can
lead to epithelial defects, which asa result of compro- mised wound
healing persist and progress.
Surgical attempts to rehabilitatethe ocular sur- face in severelydry eyes fail frequently.10 11 In thissituation it is important tolubricate the ocular surface
however, the ideal tear substi- tuteshould, in addition, provide
epitheliotrophic support.
The concept of natural tearsubstitutes
With few exceptions
pharmaceutical products are
optimised for their biomechanicalproperties only.1214 Fibronectin,
vitamins, and growth factorshave been used in vitro and in
vivo to encourage epithelial woundhealing. However, owing to stability
concerns and limited clinical success,
such single compound approaches
failed to become incorporated into
routine clinical management.1517
Serum and other bodily fluids have
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been used as natural tear substitutes. They are
applied as unpreserved, autologous products
18 19
proposed.
and thus lackantigenicity. Serum is the fluid
...........................................................................
Eye drops made from autologous serum are a
component of full blood thatremains afterclotting. It contains a large
variety of growth factors, vitamins,and immunoglobulins, some inhigher concentrations than in
natural tears (table 1).2 16 20 20a
These epitheliotrophic factorsare thought to be responsible for the therapeutic
See end ofarticle forauthorsaffiliations.......................
Correspondence to: GerdGeerling,DepartmentofOphthalmology,
University ofLu beck,RatzeburgerAllee 160, D-23538Lu beck;Germany;ggeerling@ophtha. mu-luebeck.de
Accepted for
publication11 June 2004.......................
new therapeutic approach for
ocularsurface disorders, such as
persistent epithelial defects or
severe dry eyes intractable to
conven- tional therapy. Their use
was first described by Fox et al in
1984 in their search for a tearsubstitute free ofpotentially
harmful preserva- tives.1 Later
Tsubota et al realised that becauseofthe presence of growth factors
and vitamins serum eye drops
might also have a true
epitheliotrophic potential for the
ocular surface.2
Here, we review the theoretical
background and the currently
available literature on the use of
this new approach and discuss
some legislative implications.
effect of serum observed on ocular
surface disorders.17 2123 The growth
and migration pro- moting effects
of serum on cell cultures ingeneral and on corneal epithelial cells
are well documented.24 25 Fox wasthe first to use serum
Abbreviations: BSS,balanced salt
solution; EGF, epidermal growthfactor; GvHD, graft versus hostdisease; Hb, haemoglobin; HBV,hepatitis B virus; HCV, hepatitis Cvirus; HTLV, human T cell lymphomavirus; KCS, keratoconjunctivitissicca; OSD, ocular surface disease;PED, persistent epithelial defect;RES, recurrent erosion syndrome;rpm, rounds per minute; SLK, superiorlimbal keratoconjunctivitis; SOP,standard operatingprocedures;TGF-b, transforming growth factor
beta
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Table 1 Comparison of the
biochemical properties of normal,
unstimulated human tears, and serum5152
TearsSerum
pH 7.47.4
Osmolality (SD) 298
(10) 296
EGF (ng/ml) 0.2
3.0 0.5
TGF-b (ng/ml) 210
633
Vitamin A (mg/ml) 0.02
46
Lysozyme (mg/ml) (SD) 1.4
(0.2) 6SIgA (mg/ml) (SD) 1190(904) 2
Fibronectin (mg/ml) 21205
EGF, epidermal growth factor; TGF-b,
transforming growth factorbeta; SIgA,
surface immunoglobulin A.
to treat human dry eyes. However, the recent
renaissance of this therapy began whenTsubota in 1999 described its successfuluse in eyes with persistent epithelial defects.
Experimentalstudies
Tsubota was also the first to show that
serum supports migration of an SV40transfected human corneal epithelial cellline in a dose dependent manner25 and thatimmortalised,conjunctival epithelial cells as a sign of higher differentiation
Autologous blood serum for topical use in
the eye. To be stored frozen and usedwithin 3 months after date of
production; To be discarded at the end of theday. A volume of 2 ml of the solution isas required by the European
Pharmacopoeia addendum 2000sent for
microbiological evaluation.The product is available approximately4 hours after
venesection, but is only dispatched oncenegative serology and microbiology of
donor and product are confirmed.Usually the drops are applied eight times
daily. A newbottle is opened every day. It isrecommended to be stored at +4
C
and to be discarded after 16 hours ofuse with regularhousehold waste. The remaining bottles
are stored frozen (ideally at2
20C) for upto 3 months. If the domestic freezerhas nothermometer it is recommended to placeone insideand control the temperature when taking anew vial out every day. If the temperaturecan not be adjusted to about 220
C the
dispensing doctor may considerrecommendation ofshorter storageepisodes.
SOP of theNational Blood Service in England andWales
Following the principles of good
manufacturingpractice theNational Blood Service (NBS) in Englandand Wales has
27
start to express mucin-1.2 In our own dose andtime response experiments in a definedculture model, we found that serum
maintained morphology and supportedproliferation ofprimary human corneal epithelial cells far better than
chosen a different approach.Patients are assessed for their
suitability to donate according to theBritish Committee for Standards inHaematology (BCSH) guidelines for pre-deposit autologous
28
unpreserved or preserved pharmaceutical tear substitutes.24 blood
donation.This requires them to be inreasonably good
Ebner et al recently also showed that
incubation ofprimary cultures of humankeratocytes with undiluted serum
increased transcription of RNA for nerve
growth factor (NGF) as well as TGF-b
receptors.26
PREPARATION AND
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USE The protocols to produce serum eye
drops in published reports areincomplete or vary significantly. Below
we describe our current standardoperating procedures (SOP) as well as
published modifications (fig 1, table 2).
SOP at the U niversity o fL ub eck
, GermanyPatients are assessed for their suitability todonate according to the guidelines of theBundesrztekammer and Paul EhrlichInstitute for blood donation and use of bloodproducts. This includes testing forhepatitisB (HBV) and C (HCV), syphilis, and HIV
serology (HbsAg; antibodies to HCV, HIV-I/HIV-II, HIV-NAT, syphilis; HCV-NAT)
before blood is donated for the productionof eye drops. A positive serology excludes
thepatient from the use of autologousserum eye drops. Following a thoroughinformation briefing regarding theexperimental nature, potentialcomplications, and alternative treatmentoptions, informed consent is obtained. Allsteps are documented on a standardoperation procedure form. Venesection is
performed at the antecubital fossa underaseptic conditions and 100 ml of wholeblood are collected into sterile containers.
The containers are left standing for 2hours at room temperature in anupright position to ensure completeclotting before they are spun in a
laboratory centrifuge at3000 g for 15 minutes. The supernatantserum is removedunder sterile conditions in a laminarairflow hood with sterile 50 ml disposablesyringes. Following this protocol,100 ml of whole blood will yield 3035 mlof serum. The
volume retrieved is determined, diluted1:5 with sterile balanced salt solution(BSS), and filter sterilised (0.2 mm). Gentleshaking ensures homogenisation beforeportions of2 ml are aliquoted into sterile dropper bottles.The bottles aresealed and labelled with the name, date
of birth of the patient, the date ofproduction, and the instruction
health, with no significant cardiovascularorcerebrovasculardisease, and free of bacterial infection.Anaemia (haemoglo-bin (Hb) ,11 g/dl) is arelative contraindication.
Blood is collected following the sameprocedure as forall
volunteer blood donors, except thatdonation is made into a sterile blood packwithout anticoagulant. Those patients who
are not thought suitable to donate a fullunit of blood (470 ml) may have lesser
amounts collected of 250400 ml. Routinevirology testing isperformed as forvolunteer
blood donations in the United Kingdom(HBsAg, antibodies to HCV, HIV I/II,human T cell lymphoma virus (HTLV) andsyphilis, HCV NAT).
The donation is stored at +4C for 2 days to
allow theblood to clot and the clot to retractfully. Serum is then separated from the clot
of blood by centrifugation (a fulldonationyielding approximately 200 ml of serum),following which an equivalent volume ofsterile normal saline is then added to theserum. The process up to this point is
carried out in aclosed pack system by utilising a sterileconnecting device.The diluted serum is then transferred toa laminar flow cabinet in a positive airpressure clean room where 3 ml aliquots
of serum are dispensed into sterile screwcapped glass dropper bottles labelled withthe patients details and storageinstructions. Five bottles from eachbatch are sampled and undergo bacterialculture before release. The bottles are
recapped and transferred to a blast freezerwhere the diluted serum is quickly frozento a temperature of less than 230
C. The
frozen serum is stored at this temperature,until collection by thepatient, on dry ice in a
sealed insulatedcardboard box. One full donation producesapproximately150 bottles. Instructions are given to thepatient about transferring the bottles totheir home freezer. The shelf life of the
product is stated as 6 months from thedate of manufacture. Patients areinstructed to use one bottle ofserum perday, instilling at a frequency determined
by symptoms (usually 36 times daily). Any
remaining serum and the bottle are
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discarded at the end of the day.
Vari ations ofproduction
While serum from various patients willcertainly not be iden- tical, it is also knownthat production factors significantly
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Autolo go us s erum eye drops7
Informed
consent
Serology (HBV, HCV,HIV, syphilis)
Figure 1 Standardmanufacturingprotocolfor thepreparation,storage, and use ofserum eye drops at theUniversity ofLu beck,
Germany.
Serology
positive: Patient
excluded from use
ofautologous
serum eye drops
Serology
negative:
Sterile
phlebotomy
:
100 ml, indisposablecontainers
Allow clotting for 2hours at 21C
Centrifuge for 15 minutes at 3000 g
Dilute to 20% to
100% with BSS
Filter sterilise (0.2 m)and aliquot 2 ml
portions in sterile
bottles
Check for microbial contamination
Negative: hand out to patient Positive: discard
Store
frozen
Use new bottle
daily at 4C, up to
hourly
Discard
daily
(regular
waste)
Return to
checkfor
microbial
contamination
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Autolo go us s erum eye drops8Downloaded from bjo.bmj.com onJuly 8, 2013 - Published by group.bmj.com
Geerling, MacLennan, Hartwig88
influence the biochemical properties of
blood derivedproducts (table 2). The effect
of the centrifugation depends upon the
centrifugal g force and time used tospin the sample. A centrifugation of 3000 g
for 15 minutes results in good separation ofserum and blood clot, without inducing
haemolysis (fig 2).29 At a low g force or short
centrifugation,platelet membranes remain
in the supernatant and in high
concentrations induce apoptosis. 30 It is also
important to understand that the g force
not only depends upon the
Table 2 Parameters that have to bedefined in theproduction of serum eye
drops and previously described
variations, storage, and application
Production factor Published
variations
Clotting phase 02days
Centrifugal force 1500
rpm (ca 300 g) to 4000 g
(ca
5000 rpm) Duration of
centrifugation 520
minutes
Dilution 20%,
33%, 50%, or100%
Diluent 0.9% NaCl, BSS, 0.5%
chloramphenicol eye
drops
Container 16
ml in insulin syringeordropperbottle
Storage220 to +4 C Number ofdaily applications 3 timesto hourly
rpm, rounds per minute; g, g force; BSS,balanced salt solution.
revolutions of the rotor per minute (rpm),
but also on the diameterof the rotor. Thus,
g force and not rpm should be stated in the
protocol. The g force in the studies publishedso far, ifmentioned at all, probably varies by
at least 1 log.
We recently quantified EGF and TGF-b1
in undiluted serum samples of 10patientsand found a much lower concentration ofTGF-b
1than Tsubota, who used a lower
rpmand thus probably a lower g force(table 3). As TGF-b potentially slows downepithelial wound healing, Tsubota
suggested diluting the serum 1:5 withphysiological saline. However, at the sametime this reduces the concentration ofother
growth factors, such as EGF, that are proved
to support proliferation of corneal epithelialcells.
In addition, concentration and diluent in
serum eye drops vary significantly. Daily
dosage containers are predominantly used
and discarded at the end of the day, but
dilution with chloramphenicol 0.5% which
has few toxic side effects has also beenadvocated to allow use of the dropper bottles
forup to 1 week.3133 Based on in vitro resultswe currently use 20% serum diluted in BSS.34
C L INICAL RESULTS
Serum eye drops have been used for
severe dry eyes, persistent epithelial
defects, and superior limbal keratocon-
junctivitis as well as a supportive measure in
ocular surface reconstruction. Allpublishedstudies are listed in table 4.
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Figure 2 Demonstration of the influence of
the g force on the volume of serum obtainedfrom 50 ml of full blood centrifuged 2 hoursafterdonation for 10 minutes at (left) 3000 (g)or (right) 500 (g). (Taken from Geerling et al,with permission of Springer Verlag,Heidelberg, Germany.)
Severe dry e ye and superior
limbalkeratoconjunctivi
tis
In 1984 Fox et al reported that a 3 week
course with serum diluted 1:2 with 0.9%NaCl solution was able to improve the signs
and symptoms in 30 eyes of 15 patients.1
When this medication was replaced by
serum diluted 1:200 or pure diluent thediscomfort increased again. Fifteen years
laterTsubota et al reported that symptoms,
fluorescein, and rose bengal staining of dry
eyes in Sjogrens syndrome were
significantly decreased after 4 weeks of 20%
serum eye drops
610 times daily.2 Similar findings weredescribed in patients with dry eye becauseof graft versus host disease,35 36 with
symptoms improving within days, butpunctate epithelial staining improving
only after months. In 14 patients with
moderate keratoconjunctivitis sicca (KCS)
in graft versus host disease (GvHD)
(Schirmertest ,10 mm) the improve- ment
lasted more than 6 months,but sixpatients
required additional punctal occlusion. In a
prospective study by Poon et al an
improvement of subjective and objective
criteria ofsevere dry eyes (Schirmer test ,5
mm). was found in all eyes receiving 100%
serum (n = 3), but only in three out ofeight
eyes on 50% serum.33 The efficacy may bedose dependent since in a study by
Takamura 94% ofpatients receiving eight
applications reported reduced symptomscompared to only
58% of those receiving
fourdrops.37
However, in a placebo controlled
prospective study in severe dry eye 20%
serum six times daily was not able to
improve discomfort or objective signs
significantly better
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than the diluent 0.9% saline.38 Sincefluorescein and rose bengal stainingtended to be reduced after 2 months oftreatment, the authors concluded that alarger controlled study was required.Noble et al compared the efficacy of3 months ofautologous serum 50% dilutedinphysiologicalsaline in a prospective clinical crossover trial
as a substitute for the conventional therapyalready used by each patient. 39
Ten out of 16patients improved. Impressioncytology showed improvement in nine, nochange in 10, and deterioration in six oftreated 25 eyes.
Superior limbal keratoconjunctivitis (SLK),a rare, chronic,
inflammatory disease with rose bengalstaining of the corneal and conjunctivalepithelium at the 12:00 limbus is thought toresult from a localised tear film deficiency.
In aprospective cohort study 20% serum eyedrops were used as additional therapy 10times daily for bilateral SLK. Within 4
weeks discomfort improved in nine of 11
and epitheliopathy in all patients. Tearbreak up time increased significantly
and conjunctival squamous metaplasia wasreduced. When the serum application wasdiscontinued, discomfort increased again.40
Overall, the efficacy of serum drop therapy
variesbetween
30100% forsymptomatic relief, 3961% forreduction of fluorescein, and 3368% for
rose bengal positive epithelio-pathy. These
discrepancies may result from variations inthe study populationsthat is, the degree ofaqueous deficiency, as well as fromvariations in the production and treatmentprotocol for serum eye drops. In some, serum
was used as an additive rather than a
substitute treatment and in otherstherapeutic contact lenses or punctal
occlusion were applied with initiation of
serum eye drops. Increasing fluid supplyrather than the epitheliotrophic nature ofserum may thus have yielded the
beneficial effect. Comparison of the
published data is further limited byvariations in reportingsuccess of treatment as (a) number of
patients improving or (b) mean change of aparameter.
Recurrent erosion syndromeRecurrent erosion syndrome (RES) is afrequent complication of trauma or corneal
basement membrane dystrophy leading torepeated episodes of irritation, pain,epiphora, and hyperaemia because ofinsufficient adhesion of the basal epitheliallayers to the underlying basementmembrane. In a prospective cohort study
11 patients with unilateral post- traumaticRES used unspecified unpreserved
pharmaceutical tears and 20% serum eyedrops three times daily for 3 months in atapered fashion. No comment is made asto whetherpreviously used alternativetreatments were suspended forthe time ofthe serum application. Mean recurrence ratewas reduced from 2.2 to 0.028/month offollow up (mean follow up 9.4 (SD 3.7)months). However, given the self healing
nature ofpost-traumatic RES and the factthat the duration since trauma was notspecified these data have to be viewed with
care.41
Persistent epit helial defectsPersistent epithelial defect (PED) can resultfrom rheumatoid arthritis, neurotrophickeratopathy, or dry eye.42 Tsubota in1999 and later Garcia-Jimenez reported theuse of 20% serum
Table 3 Concentration of EGF and TGF-b1
for undilutedserum inpg/ml (SD) as analysed by a sandwich ELISA
Centrifugation condition EGF TGF-b1
Geerling53 4000 g, 10 min 802 (155) 6029 (2517)Tsubota2 1500 rpm, 5 min 510 (80) 33 200 (6800)
Comparison of our data and results published by Tsubota (n = 10 each) illustrate the potential influence of g force on
the composition of theproduct.
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Table 4 Production conditions and results of studies published. Success is defined either as number of all eyes/patientsimproving or as reduction of mean baseline (as stated in the paper) of fluorescein, rose bengal positive epitheliopathy
(objective), or symptoms (subjective) score
Author Ye
Concentration/
diluent
Centrifuga
tion (g
force/
Clot
ting
Frequency/
remarks Indication
Eyes
(patien
Succ
ess
Succe
ss
Fox1 19 33%/0.9% 500 g/10 min NR 2 hourly KCS 30 RB 41% 51%;
(15)
Tsubota2 19 20%/NaCl 1500 rpm/5 NR 6106 KCS in SS 24 Fl: 55 %; RB: 34 %
Rocha35 20 33%/0.9% 500 g/10 min NR Hourly, KCS in 4 (2) 4 (4) 4 (4)
Poon33
20 50 4000 rpm 2 h 86 KCS 11 (9) Fl: 6 (11) BR: 6 (11)
Tananuvat38 20
chloramphenicol
(2200 g)/10min
NR 66 KCS 12
5 (11)
Fl: 39%; RB: 36%
Takamura37 20 20%/0.9% 3000 rpm/10 NR 486 KCS NR
33% IPC 44%
improved 20
Ogawa36 20 20%/0.9% 1500 rpm/5 NR 106 KCS in 28 Fl: 61%, RB 30 %
McDonnell47 19 100% NC/1520 min 15 12 hourly PED 1 (1) IC deposition No
Tsubota25 19 20%/NaCl 1500 rpm/5 NR 6106 PED 16 10 (16) NR
Poon33 20 50 4000 rpm 2 h 86 PED 15 9 (15) NR
De Souza42 20
chloramphenicol
(2200 g)/10min NR Hourly PED/PK 70 57 (70) NR
Garcia43 20 20%/0.9% 5000 rpm/10 NR 106 PED 11 6 (11) NR
Tsubota45 19 20%/NaCl 1500 rpm/5 NR J hourly LSC-Tx, PK 14 12 (14) NR
Poon33 20 50 4000 rpm 2 h 86 PK 2 (2) 2 (2) NR
Del Castillo41 20
chloramphenicol
(2200 g)/10min
NR 36 RES 11 8 (11); RoR: NR
Goto40 20 20%/0.9% 1500 rpm/5 NR 106 SLK 22 Fl 88%, BR91% 21%,
IPC 100% 9 (11)Noble39 20 50%/0.9% NR 48
Replacement OSD 32 IPC 9 (25) 10
Matsumoto20a 20 20%/NaCl 3000 rpm/10 NR 5106 NK 14 14 (14) NR
Note that the scale used to measure these changes as well as the baseline level varied between the studies. GvHD, graft versus host disease; IC, immune complex; IPC,
impression cytology; KCS, keratoconjunctivitis sicca; LSCTX, limbal stem cell transplautation;NK, neurotrophic keratopathy;NR, not reported;NS, not significant;
OS, ocular surface; OSD, ocular surface disease; PED, persistent epithelial defect; PK,penetrating keratoplasty; Replacement, frequency equivalent topreviously
applied pharmaceutical tear substitute; RoR, rate ofrecurrence; SLK, superior limbal keratoconjunctivitis; SS, Sjogrenssyndrome.
in a prospective case series. Manypatients also suffered from anaqueous deficient dry eye or corneal hypaesthesia. The
PEDs had persisted despite treatment with lubricants orbandage contact lenses for more than 2 weeks (mean 7.2 (SD9.4) months). Ten out of 16 defects healed completely within
4 weeks of initiation of therapy. Wound closure tended tocorrelate negatively with the duration of the defect before
therapy with serum.25 43 Poon used 50100% serumeye drops as a substitute for unpreserved
pharmaceutical lubricants. In nine of 15eyes that had had a PED for 7.5 (SD 5.8)
(range 124) weeks healing started within 2 weeksand was completedafter 3.6 (SD 2.5) weeks (range 3 days8weeks). However, six out of the nine defects
recurred when serum eye drops were
changed back to pharmaceutical
lubricants.33
In a prospective study De Souza
treated 70 epithelial defects (63 patients)with undiluted serum hourly in addition toroutine medication; 45 of these defects had
occurred early after penetratingkeratoplasty.42 With a mean time of 15 (SD
17) days, the history of PED was short; 57
defects healed within 14 (12) days, butnine (16%) recurred within2 months when serum drops were
stopped. Total mean follow up was 12 (4)
months. Neither size nor localisation of thedefect correlated significantly with success
or failure, but deeper defects showed a
tendency to heal less successfully. None ofthese studies however was placebo
controlled.Meanwhile, umbilical cord serum has
been advocated as an alternative treatmentfor promoting corneal epithelial woundhealing. In a prospective randomisedcontrolled clinical trial on 60 eyes this ledto faster healing ofPEDs refractory to all
medical management compared to auto-
logous serum. While all
considerations regarding thepreparation,
quality control, and legal implications
are also valid for this option, additionaldifficulties may be associated withobtaining and using this allogenic blood
preparation.44
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Adjunct ive t reat me nt in oc ula rsurface r
eco n st ruct ion Surgical attempts torehabilitate severe ocular surface disease(OSD) in absolute aqueous deficiencyfrequently fail. In a prospective cohortstudy, 14 eyes of 11 patients who hadreceived a limbal stem cell graft, amniotic
membrane, and/or penetrating keratoplasty
were treated with 20% serum eye drops.Because of Stevens-Johnson syndrome orocular cicatricial pemphigoid, all eyes had
Schirmer test results of
0 mm with nasal stimulation. During theshort follow up of
20 weeks, 12 of the 14 reconstructions
resulted in a stable corneal epithelium.45
Poon confirmed this in two eyes
undergoing keratoplasty for PEDs, but
epitheliopathy recurred when thetreatment was discontinued. However, in
young patients with severe OSD andabsolute dry eye surface reconstruction
failed despite the use of autologous serumeye drops.46
Co mpl i cations
The number of complications in the 255patients reported to have been treatedwith serum eye drops is small. Most
authors report no complications at all. Fox etal reported no serious complications,1 butmentioned that other users hadencountered scleral vasculitis and melting
in patients with rheumatoid arthritis.Immune complex deposition afterhourlyapplication of 100% serum and one
peripheral corneal infiltrate and ulcer
within 24 hours after initiation of serumdrops have been reported.33 47 Circulating
antibodies which are present in serum eyedrops and antibodies in the cornea could
theoretically form such immune complexdeposits in the peripheral cornea andinduce an inflammatory response. In fivepatients with dry eyes, discomfort orepitheliopathy increased with the use ofserum eye drops and three patients with anepithelial defect developed a microbial
keratitis. A temporary bacterial
conjunctivitis was reported in a total offive
cases and eyelid eczema in two.35 36 38
However, these
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complications could also have occurred in thenatural course of the disease because ofother risk factors such as presence of
rheumatoid arthritis, remaining suturematerial, tear deficiency, and use of
bandage contact lenses.
PROBLEMS , LEGAL ASPECTS , QUALITYCONTROL, AND COSTSPotential disadvantages of serum eye dropsare the limitedstability and the risk of infection arising
for patients and others handling serum.Published evidence on these issues is scanty.
Stabilit
y
Tsubota et al reported that the
concentration of growth factors, vitaminA, and fibronectin in 100% and 20% serumdiluted with NaCl stored at 4
C remained
unchanged for1 month and at 220C for at least 3 months.
2As with other
bodily fluids it should be expected that at 4C protein
concentrations decrease significantly in thecourse ofweeks.Since in developed countries access tofreezers is rarely a problem it thereforeseems preferable to store unused vials ofserum frozen to optimally preserve the
activity of epithelio- trophic factors.48
However no more detailed publishedevidence is available to clarify the minimumfreezer temperature required for severalmonths ofstorage.
Risk ofinfection
One of the disadvantages of autologousserum eye drops is the risk ofinducing aninfection because of, for example,hepatitis of the donor or microbialcontamination of the initially sterile
dropper bottle during prolonged use. Thismay occur during preparation as well asapplication of the drops either to the corrector any accidental recipient. Transmission ofHIV by a single serum droplet into one
eye has been reported at least in one case.49Although serum is known to have
antimicrobial proper-ties, so far this has not been
quantified for diluted cryopreservedserum. When used in a hospital setting
and applied by trained personnel no
contamination was observed in weekdosage dropper bottles until the fourthday.50
However, when the drops were used in a
domestic setting by the patientsthemselves microbial keratitis evolved inthree out of 13 eyes with PEDs treated
with serum and contamination of dropperbottles was observed despite using0.5% chloramphenicol as a preservative.33
Since laboratory evidence suggests thatdilution with an antibiotic may reduce theepitheliotrophic capacity of serum eye dropsand ocular surface disease often requires
long term treatment, hospita- lisation ofpatients for this purpose is not a suitableoption and storage of the serum product inday dosage vials seems preferable.
Standardising t he production of serum eye
drops Neitherproduction, nor application(that is, frequency) ofserum have been
standardised so far. It also remains unclearwhetherthe serum should be filter sterilisedto remove any corpuscularbloodcomponents. Fox recommends filtration toremove fibrin strands, suspected to reducethe effect ofserum eye drops. The variableefficacy of serum eye drops suggests that the
parameters for theirproduction should bestandar- dised and optimised before anymeaningful placebo con- trolledrandomised clinical trial can evaluatethe realpotential of this new therapy.34
Legal aspectsAutologous serum eye drops are a medical
product. The distribution ofpharmaceuticalproducts is regulated by governmental lawsin most countries. In the European
Union (EU), the manufacture anddistribution of pharma- ceuticals areregulated by the individual country;however,
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several directives have been issued (1965/65,
1975/139, 1975/
318) by the European parliament andcouncil. These directives had to be taken
into account in the laws ofeach member
state of the EU and led to the
implementation of a marketingauthorisation to be issued by acompetent authority of each state. Themarketing authorisation depends on the
proof of efficacy in clinical trials,
implementation ofquality controls, reports
of adverse effects, proof of expertknowledge, and other regulatory aspects.
These criteria can, in most cases, probablyonly be fulfilled by professional
pharmaceutical manufacturers.
An exemption from the need to obtainmarketing authorisation is granted, if a
physician manufactures or prescribes aspecific medical product to treat his ownpatient on a named basis. This product hasto be prepared according to the doctorsspecifications, whose responsibility itremains that manufacture and applicationare performed correctly. Autologous serum
eye drops can therefore be produced by thephysician himself or on his prescription.
Since stricter regulations, especially forblood products, may exist in individualstates every physician producing or
prescribing autologous serum eye drops hasto appraise himself about specific nationalregulations.
In England and Wales the NBS has
supplied autologous serum eye drops so far
under a drug exemption certificate for thepurposes of a clinical trial from the regulatory
body in the United Kingdom, the Medicines
and Healthcare Regulatory Agency(MHRA).39 The NBS hopes to be able to
offer this therapy nationally in the nearfuture, following inspection oftheprocedureby the MHRA. At this stage the treatment willcontinue to be monitored by the MHRA
who will receive annual safety reports.
In the United States, producers of
drugs and medical devices have to be
registered with the Food and Drug
Administration (FDA). Similar to EU
regulations, registration is not necessary
for practitioners licensed by law toprescribe or administer drugs or devices
and who manufacture, prepare,propagate, compound, or process drugs or
devices solely for use in the course of their
professional practice. Again, special
regulations on testing and approval of drugsby the FDA or for using bloodproducts have
to be evaluated by the practitioner.
Quality control andpatient information
Before application, the patient must beinformed in writing about the planned
therapy, its experimental nature, the risksinvolved (for example, bacterial
contamination), and alter- native methodsof treatment. The patients informedconsent should be obtained. If only a
small amount of blood (50100 ml) is taken mild anaemia or
circulatory disorders need not be
considered as contraindications. However,in order to minimise the danger of
bacterial contamination, no blood should
be taken from patients with suspectedsepticaemia, and to reduce the risk of viral
transmission to others, patients testingpositive for HIV, HBV, HCV, or syphilis
should be excluded.
Bacterial contamination is a potential riskin the produc- tion and use of serum eye
drops. Sterile manufacturing conditions,beginning with thorough skin disinfection,are ofthe utmost importance. It is preferablethat further process- ing is performed in a
closed system. A microbiological
examination of the product should be
performed before application in order to
rule out bacterial contamination resulting
from the production process. To minimisethe riskof infection to third parties (for
example, production ornursing staff) it isstrongly recommended that the donor istested for HIV, HBV, and HCV and a control
system must be implemented to ensure that
the product is only used when themicrobiological and serological tests are
clear.
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Autolo go us s erum eye drops1473
Before venesection and application, theidentity of thepatient must be confirmed.The packaging must be clearly labelled with
the patients name, date of birth, acomment that the material is an
autologous blood product, which is solelyfor application to the patient named, aswell as the name and address of the
manufacturerand the date ofmanufacture.
To minimise the variability of the productand to maximise the safety of its use, awritten version of the standard
production procedures should beestablished. Conscientious documentation
is indispensable for good medical andmanufacturing practice. Each step relevant
to manufacture as well as application(including the dates ofapplication and anyunwanted effect) should be recorded.
From this it becomes obvious that strictguidelines for good manufactur- ing, qualitycontrol and documentation must beestablished and maintained before andthroughout the therapeutic use ofautologous serum eye drops.
Cost
sAs the production of serum eye drops fromautologous blood is time consuming andlabour intensive the costs of this approachhave to be considered. Table 5 shows thecosts for the production of serum eyedrops for the pharmacy department ofthe University of Lu beck for the year2003. When excluding the costs for the
acquisition of the necessary laboratoryhardware (for example, laminarair flow
hood, centrifuge) the total costs of a daydosage is J2.27 for 20% and J4.61 for 100%serum eye drops. This is approximatelyequivalent to the costs of one bottle ofpreserved pharma- ceutical lubricant. Sinceserum eye drops are reserved forcases of
OSD refractive to other forms oftreatment,resulting in severe discomfort and/orconsiderable economic costs to society, thissum seems to be acceptable.
SUMMARY AND C ON
CLUSION
While pharmaceutical lubricants offer littleto no nutrition eye drops made fromautologous serum have a tear-like
biochemical characterand supply nutritional
components. In vitro studies have shown thatserum supports viability, proliferation andmigration of ocular surface epithelial cells
Table 5 Costs of production for 20%
and 100% serum eye drops produced out
of 100 ml fullblood and filled as2 ml aliquots in sterile dropper bottles
(calculationbased on figures of theDepartment of Pharmacy ofthe University
Hospital Lu beckfor the year2003)
Costs
(J)
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Geerling, MacLennan, Hartwig1474
better than unpreserved pharmaceuticaltear substitutes. Clinical studies describeda variable efficacy of serum eye drops in,
for example, PEDs and dry eyes refractive toconventional treatment. This in part canbe attributed to variations in themanufacturingprotocol of serum eye drops.
The production parameters should beoptimised before a meaningful randomised,controlled clinical trial attempts to evaluatethe real therapeutic potential of thisapproach in OSD. Meanwhile, the use ofserum eye drops remains an experimentalapproach. Therefore, all applicablelegislative restrictions should be carefullyconsidered and well docu- mented andinformed consent should be obtained fromeachpatient.
ACKNOWLEDGEME
NTS
This work was in parts funded by aresearch grant from theUniversity of
Lu beck.The authors thank the head pharmacist ofthe University Hospital Schleswig Holstein,
H G Strobel, for calculating the costsofproduction of serum eye drops.
.....................
Authors
affiliationsG Geerling, Department of Ophthalmology, University ofLu beck,Ratzeburger Allee 160, 23538
Lu beck,Germany
S MacLennan, National Blood Service, Leeds
Centre, Bridle Path, Leeds
LS15
7TW, UK
D Hartwig, Institute of Immunology andTransfusion Medicine, University ofLu beck,
Ratzeburger Allee 160, 23538 Lu beck,
Germany
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Autologous serum eye drops for ocular surface
disorders
G Geerling, S MacLennan and D Hartwig
Br J Ophthalmol2004 88: 1467-1474doi: 10.1136/bjo.2004.044347
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