Scalable radiotherapy data curation infrastructure for deep-learning based autosegmentation of organs-at-risk: A case study in head and neck cancer.

In this era of patient-centered, outcomes-driven and adaptive radiotherapy, deep learning is now being successfully applied to tackle imaging-related workflow bottlenecks such as autosegmentation and dose planning. These applications typically require supervised learning approaches enabled by relatively large, curated radiotherapy datasets which are highly reflective of the contemporary standard of care. However, little has been previously published describing technical infrastructure, recommendations, methods or standards for radiotherapy dataset curation in a holistic fashion. Our radiation oncology department has recently embarked on a large-scale project in partnership with an external partner to develop deep-learning-based tools to assist with our radiotherapy workflow, beginning with autosegmentation of organs-at-risk. This project will require thousands of carefully curated radiotherapy datasets comprising all body sites we routinely treat with radiotherapy. Given such a large project scope, we have approached the need for dataset curation rigorously, with an aim towards building infrastructure that is compatible with efficiency, automation and scalability. Focusing on our first use-case pertaining to head and neck cancer, we describe our developed infrastructure and novel methods applied to radiotherapy dataset curation, inclusive of personnel and workflow organization, dataset selection, expert organ-at-risk segmentation, quality assurance, patient de-identification, data archival and transfer. Over the course of approximately 13 months, our expert multidisciplinary team generated 490 curated head and neck radiotherapy datasets. This task required approximately 6000 human-expert hours in total (not including planning and infrastructure development time). This infrastructure continues to evolve and will support ongoing and future project efforts.

Utility of viscoelastography with TEG 6s to direct management of haemostasis during obstetric haemorrhage: a prospective observational study.

BACKGROUND: The TEG 6s is an automated cartridge-based device with limited description of use in obstetric haemorrhage. The aim of this analysis was to describe the utility of TEG 6s in identifying abnormal laboratory results of coagulation and platelet count, and inform an interventional treatment algorithm for postpartum haemorrhage. METHODS: A prospective observational cohort study of 521 women with moderate to severe obstetric haemorrhage (>1000 mL blood loss), including 372 women with at least one TEG 6s test. A non-pregnant control group was used for reference. TEG 6s test parameters Citrated Functional Fibrinogen (CFF), Citrated Kaolin TEG (CK) and Citrated Rapid TEG (CRT) were compared with paired laboratory tests of fibrinogen, PT/aPTT and platelet count, obtained during haemorrhage. RESULTS: Among 456 TEG 6s tests, 389 were matched with laboratory coagulation results. The receiver operator characteristic area-under-the-curve (95% CI) for CFF amplitude by 10 min to detect Clauss fibrinogen ≤2 g/L was 0.95 (0.91 to 0.99) (P<0.0001, sensitivity 0.74 and specificity 0.97 at ≤17 mm). False positives had median (IQR) Clauss fibrinogen of 2.4 (2.3-2.7) g/L. The CK-R time had some utility for detecting prolonged PT/aPTT, however a threshold for fresh frozen plasma transfusion was not established. A CRT maximum amplitude <57 mm, when CFF was ≥15 mm, identified four of eight samples with platelet count <75 × 109/L. CONCLUSION: The TEG 6s CFF can be used to identify low fibrinogen during obstetric haemorrhage. A value to identify transfusion thresholds for PT/aPTT and platelets was not established, and laboratory results should continue to be used.

Long term results of diode laser cycloablation in complex glaucoma using the Zeiss Visulas II system.

AIM: To investigate the safety and efficacy of the Zeiss Visulas II diode laser system in the reduction of intraocular pressure (IOP) in patients with complex glaucoma. METHODS: The authors analysed the medical records of patients who underwent trans-scleral diode laser cycloablation (TDC) at the Manchester Royal Eye Hospital during a 34 month period. 55 eyes of 53 patients with complex glaucoma were followed up for a period of 12-52 months (mean 23.1 months) after initial treatment with the Zeiss Visulas II diode laser system. RESULTS: Mean pretreatment IOP was 35.8 mm Hg (range 22-64 mm Hg). At the last examination, mean IOP was 17.3 mm Hg (range 0-40 mm Hg). After treatment, 45 eyes (82%) had an IOP between 5 and 22 mm Hg; in 46 eyes (84%) the preoperative IOP had been reduced by 30% or more. The mean number of treatment sessions was 1.7 (range 1-6). At the last follow up appointment, the mean number of glaucoma medications was reduced from 2.1 to 1.6 (p<0.05). In 10 eyes (18%), post-treatment visual acuity (VA) was worse than pretreatment VA by 2 or more lines. CONCLUSIONS: Treatment with the Zeiss Visulas II diode laser system can be safely repeated in order to achieve the target IOP. Treatment outcomes in this study were similar to those from previously published work using the Iris Medical Oculight SLx laser.

Missense mutations in COL8A2, the gene encoding the alpha2 chain of type VIII collagen, cause two forms of corneal endothelial dystrophy.

Corneal clarity is maintained by its endothelium, which functions abnormally in the endothelial dystrophies, leading to corneal opacification. This group of conditions includes Fuchs' endothelial dystrophy of the cornea (FECD), one of the commonest indications for corneal transplantation performed in developed countries, posterior polymorphous dystrophy (PPCD) and the congenital hereditary endothelial dystrophies (CHED). A genome-wide search of a three-generation family with early-onset FECD demonstrated significant linkage with D1S2830 (Z(max) = 3.72, theta = 0.0). Refinement of the critical region defined a 6-7 cM interval of chromosome 1p34.3-p32 within which lies the COL8A2 gene. This encodes the 703 amino acid alpha2 chain of type VIII collagen, a short-chain collagen which is a component of endothelial basement membranes and which represented a strong candidate gene. Analysis of its coding sequence defined a missense mutation (gln455lys) within the triple helical domain of the protein in this family. Mutation analysis in patients with FECD and PPCD demonstrated further missense substitutions in familial and sporadic cases of FECD as well as in a single family with PPCD. This is the first description of the molecular basis of any of the corneal endothelial dystrophies or of mutations in type VIII collagen in association with human disease. This suggests that the underlying pathogenesis of FECD and PPCD may be related to disturbance of the role of type VIII collagen in influencing the terminal differentiation of the neural crest derived corneal endothelial cell.

Developmental changes in patterns of expression of tenascin-C variants in the human cornea.

PURPOSE: To study patterns of expression of alternatively spliced tenascin-C (TN-C) variants believed to mediate cellular activities in human corneal development. METHODS: Serial sections of preterm, neonatal, child, and adult globes with normal anterior segments were labeled with monoclonal antibodies to TN-C. The antibodies included BC-4 and BC-8, which recognize epitopes in conserved domains of TN-C and can thus detect all TN-C variants, and BC-2, alpha-A2, alpha-A3, alpha-IIIB, TN11, and alpha-D, which bind to epitopes in alternatively spliced fibronectin type III repeats of TN-C. Bound antibodies were localized and visualized using an avidin-biotin complex-alkaline phosphatase technique. RESULTS: BC-4 and BC-8 showed similar patterns of staining, widely observed in preterm corneas, less so in neonatal corneas, and restricted to the limbus in the child and adult. BC-2, alpha-A2, alpha-A3, alpha-IIIB, TN11, and alpha-D staining was largely localized in corneal epithelium (preterm and neonatal), limbal epithelium, mast cells, and matrix surrounding limbal vessels (preterm, neonatal, child, and adult). CONCLUSIONS: TN-C may play a role in corneal development and in growth and differentiation of stem cells because it is widely expressed in the preterm cornea, less so in the neonate, and is restricted to the limbus in the child and adult. The differential patterns of expression of TN-C variants in normal corneas (preterm and neonatal), and in the limbus (preterm, neonatal, child, and adult), suggest specific roles played by each variant, and cell type-specific expression of the different variants.

Management of pellucid marginal corneal degeneration.

PURPOSE: A retrospective study to ascertain the management of pellucid marginal corneal degeneration (PMCD). METHOD AND RESULTS: Sixteen patients (average age 42.6 years) presented with PMCD. PMCD was bilateral in 13 and unilateral in 3 patients. Eight eyes underwent surgery. Nineteen eyes were managed non-surgically. Surgery involved corneal wedge excision (WE) (6 eyes), penetrating keratoplasty (PK) (3 eyes) and lamellar thermo-keratoplasty (LTK) (1 eye). Immediate pre-operative average visual acuity (VA) was 6/24, 6/10 and 6/60 with an average pre-operative astigmatism of 11.40 D, 9.75 D and 20.5 D for WE, PK and LTK respectively. After an average post-operative follow-up of 57 months, 66 months and 1 year, the average astigmatism was 8.90 D, 4.63 D and 6.00 D with an average final VA of 6/19, 6/15 and 6/6 for WE, PK and LTK respectively. In the nonsurgical group, at presentation, 40% of eyes had a VA of 6/12 or better. After an average follow-up period of 32.3 months, 80% of eyes had a visual acuity of 6/12 or better. Optical correction was achieved with spectacles and or contact lenses. CONCLUSIONS: Surgical correction for PMCD provides poor long-term reduction of astigmatism. Patients with PMCD may be adequately corrected in the long term by the use of scleral fitted gas-permeable contact lenses.

Ultrastructural and molecular analysis of Bowman's layer corneal dystrophies: an epithelial origin?

PURPOSE: Two mutations (R555Q and R124L) in the BIGH3 gene have been described in anterior or Bowman's layer dystrophies (CDB). The clinical, molecular, and ultrastructural findings of five families with CDB was reviewed to determine whether there is a consistent genotype:phenotype correlation. METHODS: Keratoplasty tissue from each patient was examined by light and electron microscopy (LM and EM). DNA was obtained, and exons 4 and 12 of BIGH3 were analyzed by polymerase chain reaction and single-stranded conformation polymorphism/heteroduplex analysis. Abnormally migrating products were analyzed by direct sequencing. RESULTS: In two families with type I CDB (CDBI), the R124L mutation was defined. There were light and ultrastructural features of superficial granular dystrophy and atypical banding of the "rod-shaped bodies" ultrastructurally. Patients from three families with "honeycomb" dystrophy were found to carry the R555Q mutation and had characteristic features of Bowman's dystrophy type II (CDBII). CONCLUSIONS: There is a strong genotype:phenotype correlation among CBDI (R124L) and CDBII (R555Q). LM and EM findings suggest that epithelial abnormalities may underlie the pathology of both conditions. The findings clarify the confusion over classification of the Bowman's layer dystrophies.

Centration of donor trephination in human corneal transplantation.

PURPOSE: To develop and evaluate a new method to quantify centration of the trephined donor cornea relative to the limbus. METHODS: After human donor corneas were trephined for penetrating keratoplasty, the remaining corneoscleral discs were stained and subjected to image analysis. The centration of the excised donor cornea relative to the limbus was calculated by measuring their centroids from the "captured" images. RESULTS: Fifty-two corneoscleral discs were analyzed. The average deviation from the centre was 0.32 mm (SD, 0.18 mm). Neither surgeon nor the type of trephine significantly influenced the mean centroid deviation. CONCLUSION: We have developed and evaluated a method to quantify centration of human donor cornea. In a small series, decentration did not correlate significantly with either the surgeon or the trephine.

Graft failure in human donor corneas due to transmission of herpes simplex virus.

AIM: To report the clinical consequences of contamination of human donor corneas by herpes simplex virus (HSV) in organ culture. METHODS: Two patients without previous history of ocular HSV infection underwent penetrating keratoplasty (PK), one for keratoconus and the other for Fuchs' endothelial dystrophy. One patient suffered primary graft failure while the other developed a persistent epithelial defect, ultimately resulting in graft failure. Viral culture of swabs taken from both corneas during the early postoperative period was undertaken. The failed donor corneas were examined histopathologically by immunohistochemistry (IHC) for HSV-1 antigens, transmission electron microscopy (TEM), and by polymerase chain reaction (PCR) for HSV DNA. Both failed corneas were replaced within 6 weeks of the initial surgery. The records of the fellow donor corneas were also examined for evidence of infection. RESULTS: HSV was cultured from both corneas during the early postoperative period. Histology of both donor corneas demonstrated a thickened corneal stroma with widespread necrosis of keratocytes and loss of endothelial cells. IHC showed keratocytes positive with antibodies to HSV-1 antigens. TEM demonstrated HSV-like viral particles within degenerating keratocytes. PCR performed on the failed corneal grafts was positive for HSV-1 DNA, whereas PCR performed on the excised host corneal buttons was negative in both patients. Records of the fellow donor corneas showed that one cornea was successfully transplanted into another recipient after 18 days in organ culture, whilst the other was discarded because of extensive endothelial cell necrosis noted after 15 days in organ culture. CONCLUSION: HSV within a donor cornea may cause endothelial destruction in organ culture and both primary graft failure and ulcerative keratitis after transplantation. Endothelial necrosis of a donor cornea in culture also raises the possibility of HSV infection within the fellow cornea.

Late onset lattice corneal dystrophy with systemic familial amyloidosis, amyloidosis V, in an English family.

AIMS: To establish a clinical and molecular diagnosis in a family with late onset lattice corneal dystrophy. METHODS: Linkage analysis, single strand conformation polymorphism (SSCP) analysis, and direct sequencing of genomic DNA were performed. A review of the patients' clinical symptoms and signs was undertaken. RESULTS: Linkage to chromosome 9q34 was established and a mutation in the gelsolin gene was found in affected individuals. Numerous symptoms experienced by the patients were attributable to this mutation. CONCLUSION: A diagnosis of amyloidosis type V (familial amyloidosis, Finnish type, FAF/Meretoja syndrome/gelsolin related amyloidosis) was made. This is the first case of amyloidosis type V described in the UK. This emphasises the importance of recognition of the extraocular manifestations of eye disease both in the diagnosis and management of the patient. In addition, these findings can help molecular geneticists in their search for disease-causing mutations.

Visual function after penetrating keratoplasty for keratoconus: a prospective longitudinal evaluation.

AIMS: To evaluate visual function and vision specific health status in patients undergoing penetrating keratoplasty for keratoconus. METHODS: A prospective longitudinal study measuring logMAR visual acuity, contrast sensitivity, disability glare, binocular visual field, stereoacuity, and subjective visual function (VF-14) was conducted on 18 patients with keratoconus undergoing penetrating keratoplasty (PK), including six patients who had already had PK in the fellow eye. Data were collected preoperatively and at 3, 9, and 18 months after surgery. RESULTS: Within 3 months of surgery there was significant improvement in aided visual acuity, contrast sensitivity, and stereoacuity (p<0.05); disability glare (p<0.05) no longer had a significant detrimental effect on these variables. VF-14 score improved significantly throughout the postoperative period (p<0.05). There was significant correlation of the VF-14 score with aided visual acuity, binocular visual field, and stereoacuity. Postoperative astigmatism (<4D v >4D) did not affect the VF-14 score significantly. CONCLUSIONS: There is substantial and rapid improvement in visual function and vision specific health status in keratoconic patients as a result of uncomplicated penetrating keratoplasty.

Heterogeneity in granular corneal dystrophy: identification of three causative mutations in the TGFBI (BIGH3) gene-lessons for corneal amyloidogenesis.

Six autosomal dominant corneal dystrophies are caused by mutations in the TGFBI (BIGH3) gene on chromosome 5q31: three types of lattice corneal dystrophy (LCD), including type I and type IIIA, granular, Avellino (ACD), and Reis-Bucklers. Initially an exact genotype-phenotype correlation was reported. We report three families, with differing clinical features, all presenting with "granular" corneal dystrophy. We analysed the TGFBI gene by SSCP analysis and direct sequencing in order to further assess the genotype-phenotype correlation. We describe three separate mutations in TGFBI: one novel, one initially described as causing ACD, and one previously described. The novel mutation, R124S, is at the identical position to the mutation causing LCD type I (CDL1). We review the clinical and histological phenotypes of the corneal dystrophies and hypothesize that the ability of a mutation to cause amyloid deposition depends on the location and nature of the mutation. In addition, we suggest that the classification of the granular corneal dystrophies be revised according to mutation type and that ACD should not be classified as a distinct morphological entity.

A mutation within exon 14 of the TGFBI (BIGH3) gene on chromosome 5q31 causes an asymmetric, late-onset form of lattice corneal dystrophy.

PURPOSE: Two forms of autosomal-dominant lattice corneal dystrophy (LCD), types I and IIIA, have previously been shown to be caused by different mutations within the transforming growth factor, beta-induced (TGFBI) gene. A clinical and molecular analysis of three unrelated kindreds with a clinically distinct late-onset LCD was undertaken to determine whether this phenotype is also caused by mutations within the TGFBI gene. DESIGN: Experimental study. PARTICIPANTS: Thirty-two members of three kindreds with corneal dystrophy. DNA from 100 normal control subjects was used as a control population. METHODS: Members of three kindreds with LCD were examined clinically, and blood samples were taken for DNA analysis. Mutation analysis was undertaken on all individuals for the coding region of the TGFBI gene by means of polymerase chain reaction (PCR) followed by single-stranded conformation polymorphism/heteroduplex analysis, subcloning, and sequencing. MAIN OUTCOME MEASURES: Detection of mutations within the TGFBI gene. RESULTS: Clinical examination revealed a form of LCD that was bilateral in all but one case, with onset around the fourth to fifth decade. The majority of cases showed significant asymmetry, and in one case there was evidence of onset directly after minor superficial corneal trauma. Molecular analysis in all families demonstrated sequence changes within exon 14 of the TGFBI gene on chromosome 5q31, at codon 622 in family 3, and at codon 626 in families 1 and 2, which are presumed to be responsible for the disease. CONCLUSIONS: Previously, a late-onset form of LCD, termed IIIA, was shown to be caused by a P501T mutation in exon 11 of TGFBI. The authors present the first description of mutations in exon 14 of TGFBI causing an LCD, also of late onset. Although the condition presented is morphologically and histopathologically typical of an isolated lattice dystrophy, the age of onset and clinical course is not typical of type I, III, or IIIA lattice dystrophy. This, in conjunction with recent developments in our understanding of the molecular genetics of these disorders, calls into question the usefulness and validity of the current classification of the isolated lattice dystrophies.

Deposits and proteoglycan changes in primary and recurrent granular dystrophy of the cornea.

OBJECTIVE: To investigate the origin and distribution of granular deposits in the corneas of 3 patients with granular dystrophy, 1 of whom had previously received a lamellar keratoplasty in which the granular dystrophy had recurred. METHOD: Corneal tissue from 2 patients with primary granular dystrophy (patients 1 and 2) and from a patient with recurrent granular dystrophy (patient 3) was examined. Corneal graft tissue was fixed in (1) 3% glutaraldehyde in sodium cacodylate buffer, (2) 2.5.% glutaraldehyde in sodium acetate buffer containing cuprolinic blue, and (3) 4% paraformaldehyde in phosphate-buffered saline. RESULTS: In patient 1 (aged 48 years), electron-dense granular structures were observed in epithelium, Bowman layer, and throughout the stroma. Bowman layer was absent in several places. Patient 2 (aged 78 years) showed similar features except with more deposits in the stroma. In patient 3 (aged 48 years), granular structures were heavily deposited in the epithelium; there were also some deposits in the posterior (host) stroma, some of which were associated with partially degenerated keratocytes. Bowman layer appeared normal. In all 3 patients, the intracellular or extracellular granular structures were surrounded by fine fibrillar material and abnormal proteoglycans. Electron-lucent spaces within the corneal stroma contained large quantities of abnormal proteoglycan filaments that were attached in part to collagen fibrils. CONCLUSIONS: Results from patient 3 support an epithelial origin for the deposits, presumably from keratoepithelin, aggregated with other proteins. The role of keratocytes is less clear, although the presence of deposits in the stroma of all 3 patients, some associated with keratocytes, suggests that these cells might produce granular material in addition to abnormal proteoglycans.

A mutation in the RIEG1 gene associated with Peters' anomaly.

Mutations within the RIEG1 homeobox gene on chromosome 4q25 have previously been reported in association with Rieger syndrome. We report a 3' splice site mutation within the 3rd intron of the RIEG1 gene which is associated with unilateral Peters' anomaly. The mutation is a single base substition of A to T at the invariant -2 site of the 3' splice site. Peters' anomaly, which is characterised by ocular anterior segment dysgenesis and central corneal opacification, is distinct from Rieger anomaly. This is the first description of a RIEG1 mutation associated with Peters' anomaly.

Inhibition of human immunodeficiency virus type 1 by Tat/Rev-regulated expression of cytosine deaminase, interferon alpha2, or diphtheria toxin compared with inhibition by transdominant Rev.

A retroviral vector was designed to express toxic proteins only in the presence of the HIV-1 Rev and/or Tat protein(s). The design of this vector incorporates an HIV-specific expression cassette that consists of three elements: the U3R region of the HIV-1 IIIB LTR provides the promoter and Tat-responsive element, a modified intron derived from the human c-src gene facilitates the splicing of inserted genes, and the HIV-1 RRE region enhances the transport of unspliced mRNAs. To further limit potential readthrough transcription, the expression cassette was inserted in the reverse transcriptional orientation relative to the retroviral vector LTR. Three different genes, interferon alpha2, diphtheria toxin (DT-A), and cytosine deaminase, were inserted into this vector. Tat and Rev inducibility was demonstrated directly by a >300-fold induction of interferon production and functionally by a decrease in colony-forming units when a Tat and Rev expression vector was titered on HeLa cells harboring the inducible DT-A cassette. The Tat-inducible cytosine deaminase gene was tested in the Sup-T1 T cell line and shown to inhibit HIV-1 production only when engineered cells were grown in the presence of 5-fluorocytosine. To test the ability of this system to inhibit HIV-1 infection in bulk PBL cultures, a series of transduction and challenge experiments was initiated with both the interferon and DT-A vectors. Protection against infection was documented against three HIV strains in PBLs. Last, the interferon and DT-A vectors were compared with a vector encoding a transdominant Rev protein and were shown to mediate equal or greater inhibition of HIV-1.

Tenascin-cytotactin (TN-C) variants in pseudophakic/aphakic bullous keratopathy corneas.

PURPOSE: To examine pseudophakic/aphakic bullous keratopathy (PBK/ABK) human corneas for patterns of expression of tenascincytotactin (TN-C) variants known to mediate specific cellular functions, viz. anti-adhesion (high molecular mass (M(r))) and adhesion (low/intermediate M(r)). METHODS: PBK/ABK corneas were selected to encompass only those with bullae and without inflammation, scarring or neovascularisation. Serial sections from these and normal corneas were labelled with antibodies BC-4 (recognising all TN-C variants) and BC-2 (specific for the high M(r) TN-C variant). Bound antibody was revealed with an avidin-biotin peroxidase technique. In a given pair of corneal sections, positivity with BC-4 but not BC-2 indicates localisation of low/ intermediate M(r) TN-C variants and absence of the high M(r) TN-C variant. BC-2 identifies the high M(r) variant. RESULTS: There was no immunostaining with either BC-2 or BC-4 in normal corneas except at the corneoscleral interface, where both BC-2 and BC-4 were immunolocalised. In PBK/ABK corneas, BC-2 staining was seen in 5 of 13 corneas and was restricted mainly to epithelial basement membrane (BM) overlying bullae. BC-2 did not label the stroma. BC-4 immunostaining was present in all PBK/ABK corneas and was localised in epithelial BM, both epithelial and stromal borders of bullae, pannus, endothelial BM and in oedematous stromal regions. CONCLUSIONS: TN-C variants are differentially expressed in PBK/ABK corneas. The high M(r) variant is restricted mainly to epithelial BM overlying bullae, while low/intermediate M(r) variants occur in epithelial BM, both epithelial and stromal borders of bullae, and in pannus. Given the in vitro functions of TN-C, a role for promoting epithelial dehiscence and reattachment to the substratum in PBK/ABK corneas by high and low/intermediate M(r) variants respectively is likely.

An ultrastructural investigation into proteoglycan distribution in human corneas.

PURPOSE: We report an investigation into the distribution of proteoglycans (PGs) in normal, organ-cultured and dextran-treated human corneas. METHODS: Immunogold labeling was carried out at the electron microscope level to localize keratan sulphate (KS), chondroitin sulphate (CS), and heparan sulphate (HS) PGs. RESULTS: High levels of labeling for CS was found in the epithelium, endothelium, and keratocytes, with light labelling present in the basement membranes and the corneal stroma. Labeling for HS was present in the epithelium, endothelium, and keratocytes, with intense labeling present at the endothelium/Descemet's membrane interface and the epithelium/Bowman's layer interface. Large filaments were also observed in these regions in cuprolinic blue-stained specimens. Keratan sulphate was present at high levels in the stroma and the basement membranes with low levels present within the keratocytes, epithelium, and endothelium. The pattern of KS labeling along the collagen fibrils in the stroma sometimes showed evidence of periodicity. Organ-cultured corneas had extensive collagen-free "lakes," the interior of which immunolabeled positively for KS and showed staining with cuprolinic blue. The lakes were greatly reduced in the dextran-treated samples. CONCLUSION: This investigation determined the ultrastructural distribution of KS, CS, and HS PGs in human cornea and showed that organ culture is associated with a change in distribution of stromal PGs.