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Table of Contents
ORIGINAL ARTICLE
Year : 2022  |  Volume : 16  |  Issue : 1  |  Page : 48-55

A study of visual outcome in deep anterior lamellar keratoplasty at a tertiary eye care institute


1 Department of Ophthalmology, Sarojini Devi Eye Hospital, OMC, Hyderabad, Telangana, India
2 Department of Ophthalmology, OMC, Hyderabad, Telangana, India

Date of Submission08-Jun-2021
Date of Acceptance19-Jul-2021
Date of Web Publication31-Mar-2022

Correspondence Address:
Dr. Superna Mahendra
3-6-479, Flat No. 301, Sreeman Nirantar Complex Street No. 6 Himayatnagar, Hyderabad, Telangana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijot.ijot_58_21

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  Abstract 


Introduction: Deep anterior lamellar keratoplasty is a surgical procedure for removing the corneal stroma down to Descemet's membrane. It is most useful for the treatment of corneal disease in the setting of a normally functioning endothelium. Materials and Methods: Patients attending Sarojini Devi Eye Hospital were screened. Twenty-eight eyes of 23 patients with anterior corneal pathologies and who had a best-corrected visual acuity of less than 6/60 and not improving beyond it were included in the study. Results: Conditions in which the pathology is limited to the anterior stroma and sparing the endothelium, lamellar keratoplasty as a procedure offers many advantages with fewer risks of complications. Conclusion: Corneal pathologies involving anterior stroma are better treated with DALK with fewer complications.

Keywords: Corneal transplant, keratoplasty, lamellar keratoplasty


How to cite this article:
Mahendra S, Killani SP, Pola S. A study of visual outcome in deep anterior lamellar keratoplasty at a tertiary eye care institute. Indian J Transplant 2022;16:48-55

How to cite this URL:
Mahendra S, Killani SP, Pola S. A study of visual outcome in deep anterior lamellar keratoplasty at a tertiary eye care institute. Indian J Transplant [serial online] 2022 [cited 2022 Dec 8];16:48-55. Available from: https://www.ijtonline.in/text.asp?2022/16/1/48/342438




  Introduction Top


Deep anterior lamellar keratoplasty (DALK) is a surgical procedure for removing the corneal stroma down to Descemet's membrane. It is most useful for the treatment of corneal disease in the setting of a normally functioning endothelium. Traditionally, penetrating keratoplasty (PK), which involves a full-thickness corneal graft, has been the treatment of choice for corneal stromal diseases. However, PK can be complicated by graft rejection, irregular astigmatism, and corneal opacification, thus resulting in visual impairment. DALK offers an alternative procedure that may lessen those risks because the recipient Descemet's membrane and endothelium are preserved.[1] The endothelium of the cornea is the first tissue to die after extinction of life and that explains that graft from an eye removed more than 6–8 h after death or stored more than 4–6 days, even far below ice temperature develops an endothelial edema and is not likely to remain clear if grafted to a recipient. Lt explains the larger incidence of successes in lamellar keratoplasty where endothelium does not form a part of the donor material.[2] DALK removes and replaces the pathologic corneal stroma while preserving host healthy endothelium, which eliminates the risk of endothelial graft rejection and has a reduced effect on the endothelial cell count.[3]

The most common indication for DALK is keratoconus because patients benefit the most from preserving their own endothelium.

Therefore, the indications for DALK have expanded to other pathologies such as corneal ectasia (pellucid marginal degeneration and post laser in situ keratomileusis ectasia), stromal dystrophies (lattice, macular, and granular), stromal opacities, scars, active corneal ulcer, and perforations.[4]

Objectives

  1. To study the indications for DALK
  2. To evaluate the different surgical techniques of DALK
  3. To evaluate the outcomes after DALK in terms of:


    1. Best-corrected visual acuity (BCVA)
    2. Graft survival
    3. Complications.


Corneal anatomy and pathophysiology

The cornea is the epitome of efficiently unified structure and function, providing the eye with a clear refractive surface, tensile strength, and protection from external factors. The diverse functions have been achieved with the simplicity of the design.

The cornea is transparent, avascular, and watch glass-like structure with smooth convex outer and concave inner surface. It contributes to 70% of refractive power (+43D) with a refractive index of 1.37.

The anterior surface of the cornea is elliptical because of the greater overlap of sclera above and below.

  • Horizontal – 11.75 (11–12 mm)
  • Vertical – 10.6 mm (9–11 mm) [Figure 1].
Figure 1: Arrangement of corneal stromal lamellae

Click here to view


Microscopic anatomy

Histologically, it contains five layers:

  • Epithelium
  • Bowman's membrane (anterior limiting lamina)
  • Substantial propria (stroma)
  • Descemet's membrane (posterior limiting layer)
  • Endothelium.


Keratoplasty

Keratoplasty is a surgical procedure in which abnormal host tissue is replaced by healthy donor corneal tissue.

Classification

  1. Depending on the thickness of the donor material
  2. Depending on donor material employed
  3. Miscellaneous types.


Depending on the thickness of the donor material

Full thickness (penetrating keratoplasty): Removal of full thickness of cornea and insertion of a graft of equal or slightly larger dimension

Indications

  • Optical: For the purpose of improving visual acuity
  • Therapeutic: For treatment of diseases, for removal of inflamed corneal tissue in eyes unresponsive to conventional anti-bacterial or anti-viral therapy
  • Reconstructive/tectonic: Improve the structure of the eyes with severe structural changes such as stromal thinning or descemetocele
  • Cosmetic: For improvement of the appearance of the eye.


Partial thickness (lamellar keratoplasty)

Partial thickness of cornea replaces the selectively diseased cornea leaving the healthy cornea of the patient undisturbed.

  • Types


    • Anterior lamellar
    • Posterior lamellar.


  • Anterior lamellar


    • DALK
    • Descemetic and predescemetic
    • Microkeratome-assisted DALK
    • Femtosecond-laser assisted DALK.


  • Posterior lamellar keratoplasty


    • DLEK: Deep lamellar endothelial keratoplasty
    • DSEK: Descemet's stripping endothelial keratoplasty
    • DSAEK: Descemet's stripping automated endothelial keratoplasty
    • DMEK: Descemet's membrane endothelial keratoplasty
    • Femto laser-assisted EK.


Depending on donor material employed

  • Auto keratoplasty: Graft material is obtained from the same person.
  • Homo/Allo keratoplasty: Graft material is obtained from another individual of the same species
  • Hetero/Xeno keratoplasty: Graft is obtained from another species.


Preoperative considerations and decision-making in keratoplasty

  • PK remained the procedure of choice for most corneal.
  • Surgical disease entities for many decades
  • The Eye Bank Association of America statistics from 2009 revealed that 45% of all keratoplasty procedures performed in the United States were partial thickness corneal grafts.


Preoperative assessment

  1. Contribution of the corneal pathology to the patient's visual compromise
  2. The appropriateness of surgery for the individual patient
  3. The timing of such surgery
  4. Perioperative and intraoperative considerations
  5. The preparation of the patient for the anticipated lengthy postoperative course.


Preoperative evaluation

Visual acuity

  • Tear film status
  • Intraocular pressure
  • Slit-lamp examination
  • Fundus evaluation
  • Keratometry
  • Corneal pachymetry
  • Videokeratography and Orbscan
  • Specular microscopy.


Donor criteria

  1. Age
  2. Exclusion criteria:


    • Death of unknown cause
    • Death with neurologic cause of unestablished diagnosis
    • Dementia
    • Subacute sclerosing panencephalitis
    • Congenital rubella
    • Reye's syndrome
    • Active infections of the cornea
    • Prior intraocular or anterior segment surgery
    • Septicemia
    • Intrinsic eye disease
    • Leukemia
    • HIV, HbSAg, and hep C
    • Active disseminated lymphomas.


Prerequisites for LKP

  1. Achieve the deepest possible interface to reduce scarring
  2. Achieve a posterior layer of uniform thickness
  3. Perform a smooth surface sectioning of both graft and bed
  4. Make the graft tissue of appropriate thickness
  5. Ensure good cooptation of the edges and uniform traction of the suture


    • The recipient cornea is dissected first, since the operation is unpredictable and if the eye is perforated, a full-thickness graft is necessary
    • More than one eye should be at hand because of the difficulty of dissection
    • The graft should be slightly larger than the recipient bed
    • The graft should be of uniform thickness and slightly thicker than the recipient bed.


Techniques

  • These can be divided into the following groups, depending upon the type of interface created:
  • Stroma-to-stroma interface
  • Descemet's membrane-to-Descemet's membrane interface
  • Descemet's membrane (donor)-to-stroma (recipient), as in full-thickness lamellar grafts
  • Stroma (donor)-to–Descemet's membrane recipient.


Surgical steps

  • Anesthesia
  • Preparation of host bed
  • Preparation of donor material
  • Suturing
  • Anesthesia: Surgery is done either under general or local anesthesia.


Preparation of recipient bed

  1. Trephination
  2. Lamellar dissection of the marked area.


Trephination

A trephine is a sharp cylindrical blade which when used creates a corneal circular incision. The trephine is centered on the pupillary area, and the cut is made up to the depth of 0.2–0.4 mm, depending on the depth of the corneal pathology.

Lamellar dissection of the recipient bed

The depth of dissection is determined by the position of the opacities in the diseased cornea:

  • After marking the recipient cornea, a Tooke's knife can be used to create a pocket at the desired depth and the upper lip is put on a stretch with a fine forceps
  • By a constant back and forth movement into the undissected tissue, a smooth plane can be achieved using lamellar dissectors such as Gill's/Desmarre's/Crescent knife. The dissector should be directed parallel to the bed of the stromal lamellae
  • During the whole procedure, the host bed should be kept dry to aid better visualization of the plane of dissection
  • Further any inadvertent perforation during the lamellar dissection is timely diagnosed due to the presence of aqueous leak, and adequate measures may be taken to prevent its extension and subsequent complications.


Harvesting of the donor lenticule

Donor corneal tissue can be obtained from a whole eye, donor corneal tissue, or pre-carved lyophilized tissue.

Whole eye

The donor lamella can be obtained from the whole eye that can be fixed in a gauze piece or in a Tudor Thomas stand for stabilization while performing lamellar dissection.

Corneo-scleral flaps

When using the donor cornea, it is advisable that a larger rim of the host should be left. The donor button may be tightly clamped over some wetted cotton in a King's clamp.

Precarved lyophilized tissue

A contact lens lathe was used to reshape the frozen corneal tissue, which was replaced by a corneal press. These lenticules were then frozen and cryolathed and stored in McCarey-Kaufman medium for <72 h.

Suturing of the graft

A running or interrupted suturing technique using 10-0 monofilament nylon suture may be employed for lamellar keratoplasty. Good apposition of the margins is desired since imperfect apposition can result in astigmatism.

Intraoperative medications and postoperative regime

  • Subconjunctival injection of an antibiotic (gentamicin 20 mg) and steroid (dexamethasone 4 mg) combination is given in the inferior fornix at the end of the surgery, and the patient is given pad and bandage for 24 h
  • Antibiotics: Topical antibiotics such as chloramphenicol or 0.3% tobramycin are used 4 times for 1 week postoperatively or until the epithelium has covered the graft
  • Corticosteroids: Topical steroids such as 1% prednisolone acetate
  • Lubricant: Adequate lubrication is the mainstay of the postoperative management in lamellar keratoplasty. If reepithelialization of the graft fails, bandage contact lens, or a temporary tarsorrhaphy may be required
  • Suture removal: Interrupted suture must be removed as soon as the vessels bridge the host-graft junction or at 6 months postoperatively in a vascularized cornea. Suture removal may be undertaken earlier for any suture-related problems such as loose sutures, broken sutures, and suture abscesses. Selective suture removal may be done for correcting astigmatism.


Complications of anterior lamellar keratoplasty

  • Intraoperative Complications
  • Perforation of Descemet's membrane
  • Postoperative complications.


Double anterior chamber

It occurs due to the unrecognized inadvertent perforation of the host during trephination, lamellar dissection, or suturing.

  • Folds in the diabetes mellitus (DM)
  • Delayed epithelialization
  • Stromal melting
  • Microbial keratitis.


Epithelial proliferation and ingrowths, astigmatism interface scarring recurrence of the host pathology, suboptimal visual acuity.


  Materials and Methods Top


DALK was attempted at Sarojini Devi Eye Hospital, Hyderabad, in 28 eyes of 23 patients between November 2018 and December 2020.

Patients attending Sarojini Devi Eye Hospital were screened. Twenty-eight eyes of 23 patients with anterior corneal pathologies and who had a BCVA of <6/60 and not improving beyond it were included in the study.

Specific history was asked regarding h/o eye rubbing and contact lens wear. The patients underwent a detailed preoperative examination. Endothelium was assessed by specular reflection under slit-lamp under high magnification (×40) in all possible cases.

Patients with pathology involving the endothelium and those with glaucoma and any posterior segment pathology which would affect the final visual outcome were not included in the study.

Patients with ectatic disorders underwent corneal topography. Corneal ultrasound pachymetry was done in selected cases.

Lacrimal syringing was done. Informed consent was taken from all the patients and in cases of patients younger than 18 years, it was taken from their parents.

Donor's eyes were obtained from T L Kapadia Eye bank, Sarojini Devi Eye Hospital.

The patients were started on oral ciprofloxacin 500 mg BD on the day of surgery in optical cases, and the existing medications were continued in tectonic cases.

Analgesia and anesthesia were obtained with a mixture of 2% lignocaine with 1:100000 adrenaline with hyaluronidase with facial and retrobulbar block.

Once adequate anesthesia and analgesia were obtained, bridle sutures were passed through the superior rectus muscle. Lid sutures were placed in a few cases. The recipient bed was prepared first in all cases.

The recipient bed was marked with a disposable free-hand trephine, the size ranging from a minimum of 7 mm to a maximum of 8.5 mm depending on the case. The trephine was advanced to mark only the superficial stroma.

In 16 cases, the “big bubble technique” was attempted and was successful in 12 cases. In the remaining cases, after air injection, a manual lamellar dissection was continued.

After a partial thickness trephination, a small groove along the trephination margin was made with a 11 no blade and a viscoelastic was dabbed on it. With the help of a crescent blade, stromal dissection was carried out for a depth of 60%-70%. The dissected edge of the superficial flap was held with Hoskin's forceps and dissection was carried out. After an initial dissection of the anterior stroma, a big bubble was injected into the posterior stroma in a plane between the posterior stroma and the Descemet's membrane.

An insulin syringe fitted with a 26G Needle was bent at 60° at 1/3rd distance from the tip of the needle with the bevel facing down. I cc air was drawn into the syringe. Along the trephination groove, the needle was introduced into the posterior stromal plane around 4 mm from the trephination groove. Air was injected at once and a semi-opaque white disk was observed which sometimes extended beyond the groove and into the AC through the trephination groove.

After identifying the extent of the big bubble, a stab incision was made over the bubble in the center. The nick was extended and lamellar dissection was carried out in layers until most of the stroma was removed.

In 11 cases, manual lamellar dissection was carried out. Among these in two cases, there was a macro perforation due to which surgery was converted to OKP.

Donor cornea was obtained either from the whole globe or MK medium. The donor was placed in the punch with the trephine of desirable size attached to it. The button was placed endothelium up on the Teflon block and a visco placed over it. The donor button was punched with one attempt and the punched out button carefully held with a Hoskin's forceps. The endothelial side was stained with trypan blue in some cases. The endothelium was peeled of using a cellulose sponge and a Hoskin's forceps 10/0 monofilament polyamide black, was used and 16 interrupted sutures were placed.

A subconjunctival injection of gentamicin 20 mg in 0.5 ml with 0.5 cc dexamethasone was given.

All patients were started with antibiotic steroid combination drops either chloramphenicol 0.50% and dexamethasone or tobramycin 0.3% and dexamethasone. These drops were instilled hourly for the initial 2 weeks and then tapered gradually over 3 months.

Lubricating drops at an hourly dosage and then tapered and maintained at 4–6 times a day.

In cases of epithelial defects, topical antibiotic steroid was withheld and bandage contact lens is put to the defect healed.

Systemic antibiotics were continued for 5 days postoperatively. In cases of therapeutic indications, the preexisting antibiotics were continued.

All patients were followed up every day for the first postoperative week and then 2nd, 3rd, and 4th week and then every month for a minimum of 3 months and a maximum of 1 year.

In each visit, the visual acuity, the slit-lamp examination was done with special consideration for graft host interface, clarity of graft, loose sutures, any vascularization, signs of graft rejection, and infection. Intraocular pressure was recorded.

Retinoscopy was done at the end of 6 months.

Dilated fundus evaluation done at an interval of 1 month.

Statistical analysis

Data collection was done, entered in tables and charts and Descriptive Statiscal Analysis method was used to obtain results.

Declaration of Patient consent

The patient consent has been taken for participation in the study and for publication of clinical details and images. Patients understand that the names, initials would not be published, and all standard protocols will be followed to conceal their identity.

Ethics statement

Institutional Ethics Committee, OMC ECR/300/Inst/AP/2013/RR-16 (Reg no. 18112001028D) gave approval for the study. The principles of Declaration of Helsinki were followed.


  Observation and Results Top


This prospective interventional study included 28 eyes of 23 patients. Two cases were converted to full-thickness grafts and excluded from the study.

Of the 23 patients, 13 were male and 10 were female patients [Figure 2].
Figure 2: Gender distribution

Click here to view


The mean age group of the patients was 43.6 years (16–75 years) [Figure 3].
Figure 3: Age distribution

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Among the keratoconus patients one had acute hydrops with corneal scarring.

  • Five patients had nuclear sclerosis
  • Four patients were s/p Small Incision Cataract Surgery
  • One patient had both eyes chronic dacryocystitis for which dacryocystectomy was done for both eyes.


    • One patient was diagnosed as a steroid responder and laser peripheral iridotomy was done in BE
    • Preoperative visual acuity of all patients was <20\200 (logMAR >1.00)
    • All patients were followed for a period of 6 months to a maximum of 1 year.
    • Three patients lost follow-up and two presented with graft opacification 1 year later for which they underwent full-thickness regraft.


Surgical techniques

Of 27 surgeries done big bubble technique was done in 16 cases, in which 12 were successful and rest four were converted to MLD due to micro perforation [Figure 4] and [Figure 5].
Figure 4: Lamellar

Click here to view
Figure 5: Corneal stromal dystrophy

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  • Manual Lamellar Dissection was done in 11 cases, in which two cases had macro perforation and converted to full-thickness graft [Figure 6] and [Figure 7].
Figure 6: Keratoconus: Munson's sign

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Figure 7: Intraoperative pictures DALK procedure

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Visual outcome

The visual acuity at the end of 6 months is as follows:

The mean preoperative visual acuity was 1.654 logMAR units and 0.796 logMAR units in postoperative period [Figure 8] and [Table 1], [Table 2], [Table 3].
Figure 8: Postoperative pictures after DALK procedure

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Table 1: Indications

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Table 2: Preoperative and postopertaive visual acuity

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Table 3: Complications

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Applying statistical analysis, the paired t-test, the P < 0.003 which is extremely significant.


  Discussion Top


Our study aims to evaluate the visual outcomes of DALK in various corneal pathologies limited to epithelium and stroma, noting the different techniques, its advantages and pitfalls.

The study was conducted in 23 eyes of 28 patients between November 2018 and December 2020.

According to Unal et al.,[5] Keratoconus is the most common indication of DALK. In our study, there were four cases of keratoconus.

Unal et al.,[6] stated that macular dystrophy followed by lattice dystrophy is the most common indication for DALK.

In our study, there are 10 cases (35.71%) of stromal dystrophy, two cases (7.14%) of macular dystrophy. Kawashima et al.,[7] stated that DALK is not evitable for macular dystrophy as it might show progressive decrease in the corneal endothelium postoperatively. In 28 patients, five had nuclear sclerosis.

According to Coombs et al.,[8] graft survival rate is 96.8% at 1 year, 89.9% at 3 years, and 83.5% at 5 years in lamellar keratoplasty.

The mean graft survival in our group was 85.18% at the end of 1 year. The participants in our study need a longer follow-up to observe the same.

LKP does not impose stringent quality requirements on the donor graft. According to Sharma et al.,[6] this offers an advantage in a developing country like India, where tissue availability is limited, and hence LKP would help in effective utilization of the available tissue.

According to Coombes et al.,[9] some postoperative complications are comparable between DALK and PK. DALK largely eliminates some complications commonly observed after PK, such as wound leakage and endothelial graft rejection reactions.

According to Jhanji et al., tears or perforations occur in approximately 10%–30% of cases. Risk is less in elderly patients who tend to have a thicker DM.

Which correlated well with our study in which 29.41% of cases had perforations.

The three cases in which micro perforation occurred which was managed by doing manual lamellar dissection Two cases in which macro perforation of DM occurred, and these were converted to full-thickness graft.

In our study group, there was one case who presented with shallow AC after 3 months of postoperative period along with a loose suture at 4 0'clock.

She was managed by loose suture removal and AC reformation after applying glue at the suture site.

In our study, there were four cases with interface haze 10 months postoperatively which dropped the visual acuity by two Snellen lines.

This emphasizes the importance of a clear interface in the final visual outcome.

Watson, Stephanie et al. stated that epithelial, stromal, and mixed epithelial, and stromal graft rejection can occur at a rate of approximately 8%–10% which correlated well with our study.

In our study, three cases (10.75%) presented with signs of graft rejection; two cases had graft failure.

One case presented with signs at 7 months postoperative period after losing follow-up from 2 weeks postoperatively. Both were managed by regrafting.

The mean preoperative visual acuity in our study was 1.654 logMAR units and 0.796 logMAR units are postoperative BCVA.

The P < 0.003% which is extremely significant.

Persistent myopia and astigmatism following complete suture removal are the most common comorbidities which are the same in our study managed by contact lens trial.


  Conclusion Top


Conditions in which the pathology is limited to the anterior stroma and sparing the endothelium, lamellar keratoplasty as a procedure offers many advantages with fewer risks of complications.

In younger patients requiring keratoplasty for optical indications, lamellar keratoplasty is a better procedure compared to PK in preserving the patient's own functional endothelium. Donor quality is not a limiting factor in LKP, and hence allows for effective utilization of donor tissue.

The major limiting factors affecting visual outcome are graft host interface haze and irregular astigmatism.

Innovative techniques of lamellar keratoplasty give encouraging results in challenging cases which were not amenable to surgery till now.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
John V, Goins KM, Afshari NA. Ophthalmic Pearls: Cornea; Deep Anterior Lamellar Keratoplasty Eye Net Magazine; September 2007; American Academy of Ophthalmology. Available from: https://www.aao.org/publications/eyenet/200709/pearls.cfm. [Last accessed on 2021 May 15].  Back to cited text no. 1
    
2.
Dhanda RP. Keratoplasty – Recent experiences. Indian J Ophthalmol 1961;9:43-50.  Back to cited text no. 2
  [Full text]  
3.
Morris E, Kirwan JF, Sujatha S, Rostron CK. Corneal endothelial specular microscopy following deep lamellar keratoplasty with lyophilised tissue. Eye (Lond) 1998;12:619-22.  Back to cited text no. 3
    
4.
Karimian F, Feizi S. Deep anterior lamellar keratoplasty: Indications, surgical techniques and complications. Middle East Afr J Ophthalmol 2010;17:28-37.  Back to cited text no. 4
[PUBMED]  [Full text]  
5.
Unal M, Arslan OS, Atalay E, Mangan MS, Bilgin AB. Deep anterior lamellar keratoplasty for the treatment of stromal corneal dystrophies. Cornea 2012.  Back to cited text no. 5
    
6.
Ogawa A, Yamaguchi T, Mitamura H, Tomida D, Shimazaki-Den S, Murat D, et al. Aetiology-specific comparison of long-term outcome of deep anterior lamellar keratoplasty for corneal diseases. Br J Ophthalmol 2016;100:1176-82.  Back to cited text no. 6
    
7.
Kawashima M, Kawakita T, Den S, Shimmura S, Tsubota K, Shimazaki J. Comparison of deep lamellar keratoplasty and penetrating keratoplasty for lattice and macular corneal dystrophies. Am J Ophthalmol 2006;142:304-9.  Back to cited text no. 7
    
8.
Coombes AG, Kirwan JF, Rostron CK. Deep lamellar keratoplasty with lyophilised tissue in the management of keratoconus. Br J Ophthalmol 2001;85:788-91.  Back to cited text no. 8
    
9.
Sharma N, Arora T, Singhal D, Maharana PK, Garg P, Nagpal R, et al. Procurement, storage and utilization trends of eye banks in India. Indian J Ophthalmol 2019;67:1056-9.  Back to cited text no. 9
[PUBMED]  [Full text]  


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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