[Amniotic membrane transplantation in proven ulcerative herpetic keratitis: successful anti-inflammatory treatment in time]. / Amnionmembrantransplantation bei gesicherter ulzerativer Herpeskeratitis: Erfolgreiche Entzündungshemmung zum richtigen Zeitpunkt.

BACKGROUND: In active herpetic keratitis, treatment with systemic and topical aciclovir together with low dosed-out steroids has been successful for few decades. However, in cases with severe herpetic ulcer and melting inflammation, emergency penetrating keratoplasty is necessary, which carries high risk for recurrent herpetic keratitis and rejection. The anti-inflammatory effect of amniotic membrane transplantation can be used as a first surgical step in order to gain time for planned penetrating keratoplasty. The results are analysed in this retrospective case series. PATIENTS: Twelve eyes of 12 patients with clinically highly active herpetic ulcer without healing despite topical and systemic aciclovir were treated with amniotic membrane transplantation. Ten of 12 eyes (83 %) showed positive PCR testing for HSV-1. In one case, the molecular genetic result was negative, one analysis for HSV was not done. For surgery, debridement of the margins and the ground of the ulcer was done with steam heated cauterization. Afterwards, an amniotic membrane was made to cover the defect and fixed with single sutures. Peri- and postoperatively, the patients were treated with systemic aciclovir (4000 mg daily one week, 2000 mg daily 2 weeks) and gentamicin ointment topically. RESULTS: Mean follow-up was 6.8 months (1-12.5). After 25 days in the mean (11-34), nine of 12 patients experienced an intact epithelial corneal surface together with a clinically clear reduction of the inflammation. Three other patients did not recover and were treated with emergency penetrating keratoplasty after 31 days in mean (25-35). Visual acuity did not improve after amniotic membrane transplantation except in one case. CONCLUSION: The known anti-inflammatory potential of amniotic membrane causes a reduction of the inflammatory process in herpetic ulcer. A precondition is a sufficient antiherpetic and anti-inflammatory therapy and a decision for amniotic membrane transplantation in time in order to avoid an emergency penetrating keratoplasty. This gives the opportunity to perform planned penetrating keratoplasty with optimal HLA-match and postoperative immunosuppression.

[Amniotic membrane transplantation and high-dose systemic cyclosporin A (Sandimmun optoral) for Mooren's ulcer]. / Amnionmembrantransplantation und hoch dosiertes systemisches Ciclosporin A (Sandimmun optoral) bei Ulkus Mooren.

BACKGROUND: Mooren's ulcer is a rare, severe corneal autoimmune inflammation leading to blindness if treated insufficiently. High-dose systemic cyclosporin A (Sandimmun optoral) was shown to markedly reduce inflammation and stop corneal destruction. We report on three cases in which this immunosuppressive regimen required additional AM transplantation for complete healing. PATIENTS: One 37-year-old male (M) and two 49- (F1) and 58-year-old females (F2) presented with unilateral Mooren's ulcer in different stages of the disease, which deteriorated despite high-dose systemic cyclosporin A (Sandimmun optoral M, F1) or treatment with topical cyclosporin A 2 % (F2). After surgical removal of all grossly affected corneal stroma, amniotic membrane was made to cover the entire cornea and fixed with episcleral sutures in two patients (M, F2). In one patient (F1) a deep marginal ulcer was covered with a fitted AM glap. All patients were treated postoperatively up to 6 months with high-dose systemic cyclosporin A (Sandimmun optoral). Blood trough levels aimed at 150 to 200 ng/mL. Topical cyclosporin A was administered in addition in two patients (M, F2) postoperatively for at least 6 months. Due to incompatibility, one patient (F1) was treated with topical steroids instead. RESULTS: Follow-up time was 42 months (M), 50 months (F1) and 54 months (F2). All three eyes exhibited clinical healing with stable corneal surfaces thereafter. Depending on the stage of Mooren's ulcer at the time of surgery, visual acuity remained at hand motions in one patient (M) and recovered to 1.0 and 0.6, respectively, in two patients (F1, F2). CONCLUSION: Due to its anti-inflammatory potential, coverage by AM seems to trigger a therapeutic turnaround in cases of Mooren's ulcer which do not heal with intensive immunosuppressive regimens alone. In order to maintain or restore as much visual function as possible, additional amniotic membrane surgery should be performed early enough in the course of the disease.

[Significance of immediate affixation of a hard contact lens in the emergency treatment of severe alkali burns of the cornea (case report)]. / Die Bedeutung der frühzeitigen Aufklebung harter Kontaktlinsen in der Notfallbehandlung schwerer Alkaliverätzungen der Hornhaut (Fallbericht).

BACKGROUND: Severe alkali burns lead to massive limbal stem cell damage resulting in persistent epithelial defects, infiltration and stromal melting early in the disease process. A glued-on hard contact lens may serve as an "artificial epithelium" and protect the cornea from these complications. CASE REPORT: A 39-year-old male presented with severe lime burns in both eyes one week after injury. The right eye showed a totally denuded cornea and conjunctiva with circular paralimbic ischemia. In the left eye "only" two thirds of the cornea and adjacent conjunctiva and limbus were affected with less ischemia. Amniotic membrane transplantation was performed in both eyes but failed after four days already in the right eye. A hard contact lens was therefore glued on the right eye and allowed for visual acuity of 0.4 without correction in this primarily most heavily afflicted eye for 12 months duration. After removal of the contact lens, reepithelisation was quick, and function remained stable with addition of autologous serum eye drops. The primarily far less injured left eye, however, for which the amniotic membrane surgery was primarily judged to be adequate, could not be stabilised. The cornea perforated 4 weeks later necessitating a mini-keratoplasty plus a glued-on contact lens, and correctable vision never became better than 1/35. CONCLUSION: The clinical course confirms former observations that a glued-on hard contact lens is an effective treatment early after alkali burn and prevents the cornea from infiltration and melting. Moreover, two aspects merit consideration: first, with proper technique and timing of the gluing-on a patient may retain useful vision throughout the period of wearing the contact lens; second, for the long period during which the cornea was sealed by the contact lens, improper repopulation by conjunctival epithelium was inhibited, and proper corneal epithelium was given a chance for repopulation after recovery of the limbal area resulting in a stable surface and useful vision without additional keratoplasty measures.

[Ranking of systemic steroids after normal-risk keratoplasty. Results of a randomized prospective study]. / Stellenwert der systemischen Steroidtherapie nach Normalrisikokeratoplastik. Eine randomisierte prospektive Pilotstudie.

BACKGROUND: The aim of this study was to evaluate the efficacy of sole application of topical steroids after normal-risk keratoplasty. PATIENTS AND METHODS: This randomized prospective clinical study assessed 40 patients who had undergone penetrating normal-risk keratoplasty. Twenty patients were treated exclusively with prednisolone acetate 1% eye drops 5x/day for 6 months postoperatively. Another 20 patients additionally received systemic fluocortolone 1 mg/kg body weight per day tapered within 3 weeks postoperatively. The main outcome measures included clear graft survival, ratio of graft rejection, and side effects. RESULTS: The mean postoperative follow-up was 18+/-9 months. Three graft rejections were observed in the group receiving only topical steroids. Two graft rejections were observed in the group administered combined systemic and topical steroid therapy. None of the patients has developed irreversible graft failure so far. CONCLUSION: Sole topical steroid application seems to be an effective immune prophylaxis in patients undergoing penetrating normal-risk keratoplasty.

[Posterior chamber lens implantation after penetrating keratoplasty. Is this partly responsible for late transplantation failure?]. / Hinterkammerlinsenimplantation nach perforierender Keratoplastik. Mitursache für das späte Transplantatversagen?

BACKGROUND: Posterior chamber lens implantation (PCL) after penetrating keratoplasty (pKp) might contribute to late endothelial graft failure due to intraoperative damage of graft endothelium or due to PCL-associated facilitation of chronic endothelial cell loss. PATIENTS AND METHODS: A total of 55 eyes were retrospectively selected. By means of exponential regression analysis, individual annual relative endothelial cell loss was calculated. Six patients were pseudophakic prior to pKp (group A). Due to advanced cataracts in 18 patients (group B) a combined operation (triple-procedure) had been initially performed. Of the remaining 31 eyes. 17 underwent PCL implantation after a mean of 1.5+/-1.2 years post pKp during follow-up (group C). The remaining 14 eyes remained phakic during follow-up (group D). RESULTS: A statistically significant difference between the groups was not observed for the follow-up period or duration of culture ( p=0.14; Kruskal-Wallis-Test). Graft failures during follow-up were also not observed. CONCLUSION: From these preliminary results, cataract surgery does not seem to promote late endothelial failure after pKp.

Endotoxin in storage medium of human corneal grafts and clinical course after penetrating normal-risk keratoplasty.

PURPOSE: It is well known that endotoxins in storage medium may stimulate cytokine production and expression of adhesion molecules as well as endothelial damage in human corneal grafts. It has been supposed that endotoxin exposure of corneal grafts may, therefore, cause immune reactions and lead to reduced endothelial cell count after penetrating keratoplasty. It was the purpose of this prospective study to evaluate if this hypothesis is true. METHODS: A consecutive series of 274 samples of sterile organ culture storage medium from 274 human corneal grafts was collected between August 1998 and February 1999 and tested for endotoxin using Limulus amebocyte-lysate assay (LAL) after 7 days of organ culture. Threshold endotoxin level was set at 1.0 U/ml. A total of 161 grafts were transplanted and 113 were discarded. Within the 161 corneas transplanted, 62 were grafted to normal-risk patients and 99 to high-risk patients. Only normal-risk keratoplasty patients were included in the study and followed for at least 10 months. Immune reactions, graft failures, and postoperative endothelial cell counts were recorded. RESULTS: The mean endotoxin level in organ culture medium of all transplanted grafts was 1.07+/-2.96. Mean endotoxin level in organ culture medium of discarded grafts was 1.68+/-5.76, with 71 samples being below and 42 above the threshold of 1.0 U/ml called endotoxin-negative and endotoxin-positive, respectively. In all 36 culture medium samples from the 62 grafts transplanted to the group of normal-risk keratoplasty patients were endotoxin-negative and 26 endotoxin-positive. An influence of endotoxin levels on incidence of immune reactions, graft failure, and postoperative endothelial cell counts could not be revealed in patients with normal-risk keratoplasty. CONCLUSION: Low endotoxin levels in storage medium neither seem to promote immune reactions nor to contribute to postoperative chronic endothelial cell loss in normal-risk keratoplasty patients.

Detection of oestrogen receptors (ER) alpha and beta in conjunctiva, lacrimal gland, and tarsal plates.

PURPOSE: Lack of Oestrogen and androgen may be of importance in the pathogenesis of keratoconjunctivitis sicca (KCS). The signal of Oestrogens is transmitted via specific Oestrogen receptors (ER). It was the purpose of this study to evaluate the expression of ER alpha and ER beta in tear-producing tissues. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR, ER alpha + beta) and immunohistochemical evaluation (ER alpha only) were performed for ER detection and localization in tissue samples of bulbar conjunctiva (20 samples of 20 patients undergoing cataract surgery), tarsal plates (14 samples of 12 patients undergoing eye lid surgery), and lacrimal glands (20 samples of 13 cornea donors). RESULTS: Messenger RNA ER alpha was identified via RT-PCR in all tissue samples with variable expression, ER beta predominantly in lacrimal gland tissue. Immunohistochemical staining for ER alpha was negative in most cases, probably due to the thermolability of ERs and very small sample sizes. CONCLUSIONS: The detection of ER alpha and ER beta mRNA expression supports the concept of a receptor-based effect of Oestrogen in these tissues contributing to KCS. This may encourage therapeutical efforts including topical Oestrogen administration.

Organ-cultured corneal grafts from septic donors: a retrospective study.

PURPOSE: To evaluate the quality of corneal grafts from donors, who have died from septic multi-organ failure and who are called septic donors in the following. METHODS: One hundred and eighty-two corneal grafts from septic donors were stored in organ culture for 10-14 days. Graft evaluation was performed according to the criteria of the European Eye Bank Association. Only donor corneas with cell density values above 2000 cells/mm(2) were transplanted. Ninety-one patients who received these transplanted corneas were examined retrospectively with special emphasis on endophthalmitis, graft failure and incidence of immune reactions. RESULTS: Ninety-one of 182 donor corneas (50%) from septic donors were discarded mainly due to endothelial damage (61; 67%). Only seven (8%) were discarded due to medium contamination. In contrast, 452 of 1261 donor corneas (36%) from non-septic donors during the same period were discarded, again mainly due to endothelial damage (264; 58%). In this group, 48 donor corneas (11%) were discarded due to medium contamination. No patient who had received a graft from a septic donor has experienced endophthalmitis. The rate of immune reactions and graft failure was in the same range when compared to a larger group who received grafts from non-septic donors. CONCLUSION: Our data reveal no contraindication against the use of corneal grafts derived from septic donors, critical graft assessment in organ culture provided.

[Possibilities and prospects of therapy and prevention of corneal dystrophies]. / Möglichkeiten und Perspektiven der Therapie und Prophylaxe von Hornhautdystrophien.

Therapeutic approaches in corneal dystrophies should aim at long-term avoidance of recurrences. At the moment, this goal can only be achieved by transplantation of healthy corneal cells. In dystrophies of keratocytes and/or endothelial cells this can be realized by conventional penetrating keratoplasty. In epithelial dystrophies, however, simultaneous transplantation of limbal stem cells is necessary. Optimal HLA-matching strategies and immunosuppressive medication are major means to prevent immunological destruction of these.

[Perforating keratoplasty with transplants from pseudophakic donors]. / Perforierende Keratoplastik mit Transplantaten pseudophaker Spender.

PURPOSE: Pseudophacia is considered to be a relative exclusion criterion for cornea donation. The aim of this retrospective study was to investigate the clinical outcome and prognosis of penetrating keratoplasty with grafts from pseudophacic donors. PATIENTS AND METHODS: From April 1996 to July 2000 16 patients received grafts from pseudophacic donors (group I). The control group (group II) comprised 26 patients with grafts from donors without a history of intraocular surgery. Graft survival, ratio of grafts without immune reactions and chronic endothelial cell loss were compared between both groups. RESULTS: During a follow-up of almost 1 year, no graft failures and no graft rejections were observed in group I. In group II 90% of the grafts remained clear and 91% of the grafts remained free of immune reactions in the same follow-up period (Kaplan-Meier estimation). The differences between the study groups were not statistically significant. Chronic endothelial cell loss was 0.86 +/- 0.82 cells per mm2 and day in group I and 1.4 +/- 1.8 cells per mm2 and day in group II. This difference was not statistically significant. CONCLUSION: Our clinical results reveal no reason to regard pseudophacia as a principal contraindication for cornea donation, provided a critical endothelial evaluation is carried out.

Coadministration of the new macrolide immunosuppressant RAD and mycophenolate mofetil in experimental corneal transplantation.

INTRODUCTION: The effect of RAD, a new macrolide immunosuppressant, was examined as mono- and combination therapy with mycophenolate mofetil (MMF) in prevention of acute allograft rejection in murine corneal transplantation. METHODS: Both drugs were administered orally for 18 days beginning at the day of transplantation. The inbred strains Fisher and Lewis were used as donors and recipients, respectively. Five groups were involved: syngeneic control, allogeneic control, 2.5 mg/kg RAD, 40 mg/kg MMF, and double drug therapy with 1.5 mg/kg RAD and 20 mg/kg MMF. RESULTS: The median transplant survival time in the allogeneic combination was 12 (+/-0.3) days. Monotherapy with 2.5 mg/kg RAD and 40 mg/kg MMF led to a statistically significant prolongation of transplant survival to 25.5 (+/-12.5, P=0.0001) days and 19.5 (+/-13.9, P=0.0053) days, respectively. Combination therapy was superior to both monotherapies (100+/-15.8 days, P=0.03). There was a significant reduction in the number of CD4+, CD8+, as well as CD45RA+ cells in the RAD- and double drug-treated animals when compared with the allogeneic control. This significant reduction in graft-infiltrating lymphocytes has not been found in the MMF monotherapy. CONCLUSIONS: The unique finding of this first study on the combination of RAD and MMF in murine corneal transplantation is that double drug therapy produces a highly synergistic effect in prevention of acute allograft rejection without a higher incidence of complications related to drug toxicity or overimmunosuppression.

Beneficial effect of preoperative mycophenolate mofetil in murine corneal transplantation.

To investigate the effect of preoperative mycophenolate mofetil (MMF) on allograft survival in a murine corneal transplantation model. Corneal grafting was performed from Brown Norway to Lewis rats. Groups were divided as follows: Rats that received syngeneic or allogeneic grafts without therapy served as controls. MMF treatment was either started 7 days prior to transplantation and continued for 14 postoperative days (POD) or started at the day of corneal grafting until POD 14. MMF (20 mg/kg) administered postoperatively had no significant beneficial effect on corneal graft survival when compared with controls. However, the group receiving 40 mg/kg MMF postoperatively showed a statistically significant prolonged graft survival. A 1-week preoperative administration of 20 mg/kg MMF allowed superior graft survival. Priming the immune system of corneal transplant recipients preoperatively with MMF proved to be a beneficial therapeutic regimen for prolonging corneal allograft survival in rats.

Homologous penetrating central limbokeratoplasty in granular and lattice corneal dystrophy.

PURPOSE: Subepithelial recurrences in the graft are well known after conventional penetrating keratoplasties in granular and lattice dystrophies. In contrast to what is still written in most textbooks, both dystrophies have their origin in the epithelium, which originates from the limbal stem cells. The aim of this study, therefore, is to investigate whether recurrences can be avoided or minimized by simultaneous transplantation of healthy donor limbus by means of a homologous central limbokeratoplasty. METHODS: Since October 1995, five patients with granular and four patients with lattice dystrophies were treated with homologous penetrating central limbokeratoplasties. Approximately 40% of the graft's circumference contained limbal donor stem cells. After surgery, cyclosporin A was administered for at least 6 months to protect the limbal stem cells from immunologic destruction. RESULTS: During a follow-up period of 12-35 months, one recurrence was observed, probably because of an immune reaction against the transplanted limbal stem cells after tapering off the immune prophylaxis. CONCLUSION: These first results are in agreement with our expectations. More patients, however, must be operated on in this way. Follow-up must occur for many years before we will definitely know under which conditions long-term survival of donor stem cells occurs to such an extent that subepithelial recurrences in granular and lattice dystrophies stop being a major problem after keratoplasty in these patients.

Mycophenolate mofetil versus cyclosporin A in high risk keratoplasty patients: a prospectively randomised clinical trial.

BACKGROUND/AIMS: The requirement for an effective, minimally toxic immunosuppressive agent remains a major obstacle to performing high risk corneal transplantation. Although therapy with cyclosporin A (CSA) allows superior graft survival, its use is limited because of a wide range of side effects. Mycophenolate mofetil (MMF) has been shown to be a safe and effective immunosuppressive agent following renal transplantation. This prospective, randomised clinical trial was carried out to investigate the efficacy and safety of MMF in preventing corneal allograft rejection. METHODS: Recipients of corneal transplants who were at high risk for graft failure were randomly assigned to either CSA or MMF immunosuppressive therapy. CSA was given in doses to achieve whole blood trough levels of 120-150 ng/ml. MMF was given in a daily dose of 2 g. Both therapy groups additionally received oral corticosteroids (fluocortolone 1 mg/kg) which were tapered and discontinued within the first 3 postoperative weeks. Patients were monitored closely for evidence of corneal graft rejection and adverse side effects. Drug efficacy was measured, primarily, by the number of patients who experienced at least one episode of clinical graft rejection. Safety analysis focused on reported adverse side effects and laboratory measurements. RESULTS: 41 patients were enrolled in the study. There was no statistically significant difference between the two groups. 20 patients received CSA and 21 patients received MMF. Two patients in each group showed evidence of acute graft rejection which could be treated effectively by corticosteroids. All corneal grafts remained clear throughout the follow up. CONCLUSIONS: In this study it was shown that MMF is just as effective as CSA in preventing acute rejection following high risk corneal transplantation. Mycophenolate mofetil represents a promising alternative therapeutic option in patients who are at high risk for corneal graft failure.

[Endothelial evaluation of corneal transplants by digital imaging]. / Wissensbasierte Bildanalyse des Endothels von Hornhauttransplantaten.

BACKGROUND: Evaluation of corneal graft endothelium in organ culture comprises judgement of cell area, cell form and cell density by phase contrast microscopy after cell border swelling in hypotonic solution. Up to now, cell density has been determined via counting cells by hand within a fixed frame on polaroid images regarding the magnification factor. It was the purpose of this paper to establish a facilitated and reliable method for endothelial cell count by digital imaging and to compare it with our standard procedure of endothelial cell count by hand. MATERIAL AND METHODS: After cell border swelling in hypotonic solution endothelial images are recorded by a digital camera that is connected to a computer system where the grey scale images are processed into binary images using specialized enhancement and thresholding techniques. A fuzzy logic based computer program was developed in order to differentiate between single and multiple objects, i.e. living and necrotic cells. This digital imaging system determines endothelial cell density automatically and offers the possibility to correct the data in any case of misinterpretation. Twenty corneal grafts were evaluated with this system by two experienced investigators and the results were compared with endothelial cell count by hand. RESULTS: Using the new system, intra- and interindividual variability of endothelial cell count was statistically significantly lower compared with evaluation by hand. Furthermore, less time for evaluation and documentation was necessary. CONCLUSIONS: In a reduced period of time endothelial cell density of corneal grafts in organ culture can be determined reliably and reproducibly with the new digital imaging system if the possibility of data correction is used in case of misinterpretation. In the future, the necessity of corrections should be reduced and the use of multiple fixed frames should further improve quality control of corneal grafts.

Homologous penetrating central limbo-keratoplasty (HPCLK) in bilateral limbal stem cell insufficiency.

PURPOSE: Bilateral stem cell deficiency can be overcome only by keratoplasty plus additional homologous limbal transplantation. We have four years' experience with a new surgical one-stage procedure, homologous penetrating central limbo-keratoplasty (HPCLK). METHODS: A clinical trial was performed in order to evaluate the effectiveness of this new method. The first 25 eyes after HPCLK for limbal stem cell deficiency have been followed for more than 12 months. The eccentrically trephined unmatched grafts contained 40% limbus and were transplanted centrally in the host. Systemic cyclosporin A (CSA) was administered for at least one year. Central clear graft survival was the main outcome criterion. RESULTS: 18 grafts failed, mostly because of postoperative surface disorders. Seven grafts have remained centrally clear 12-41 months after HPCLK. CONCLUSIONS: In the majority of the grafts the transplanted limbal stem cells underwent immune destruction. The survival of seven grafts, however, shows that HPCLK is principally a promising new procedure. Further progress can be expected from the use of well HLA-matched grafts instead of unmatched grafts and from further improved systemic immune modulation.

[Corneoscleral transplant excision in the cadaver. Experiences of the North Rhine Westphalia Lions Cornea Bank 1995 and 1996]. / Korneosklerale Transplantatentnahme an der Leiche. Erfahrungen der Lions-Hornhautbank Nordrhein-Westfalen in den Jahren 1995 und 1996.

BACKGROUND: Donor corneas are normally obtained by whole globe enucleation-a procedure often refused by the bereaved. To increase the acceptance of cornea donation, we have exclusively obtained donor corneas by in situ excision since the end of 1994. There have been reports of increased endothelial damage and higher contamination rates. We report our experience in 1995 and 1996. METHODS: The in situ excision was performed by staff trained in microsurgical techniques. Only donor corneas with negative end-storage cultures after at least 10 days and an endothelial cell count of more than 2500 cells/mm2 were used for transplantation. RESULTS: In all, 705 corneoscleral buttons were excised from 1/95 to 12/96. The bereaved consented in 34% in 1996. A total of 30.5% of the corneas were ineligible for transplantation which corresponds to the discard figures from all cornea banks with culture methods. We did not observe any primary transplant failure nor endophthalmitis after 444 perforating keratoplasties. CONCLUSION: In situ corneal excision is safe, and helps to reduce the shortage in donor corneas.

DNA cell-cycle analysis of cervical cancer by flow cytometry using simultaneous cytokeratin labelling for identification of tumour cells.

DNA ploidy and cell-cycle distribution were determined by flow cytometry in fresh tumour tissue of 53 cervical carcinomas. Epithelial cells were labelled by a fluorescein-isothiocyanate-conjugated cytokeratin antibody (CK6, CK18) to study the influence of contaminating stromal and inflammatory cells on results of cell-cycle analysis of tumour cells. Without identification of cytokeratin-positive cells 30/53 (57%) tumours were found to be DNA-aneuploid compared to 43/53 (81%) after gating for cytokeratin. Only 7 of 15 DNA-multiploid tumours could be detected without cytokeratin staining. In addition, cytokeratin-negative cells, which are found in all tumours, can be used as an internal standard for the calculation of ploidy and for quality control (coefficient of variation, linearity) of each individual sample. Cell-cycle analysis revealed significantly higher S-phase and G2M-phase fractions in cytokeratin-gated compared to ungated samples (13.1% versus 10.0% and 8.0% versus 5.4%; P < 0.001). This difference was more pronounced in DNA-diploid than DNA-aneuploid tumours. In conclusion, about 30% of DNA-aneuploid tumours could only be detected after cytokeratin labelling of epithelial cells. Owing to the identification of cytokeratin-positive cells the influence of non-tumoural cell elements on cell-cycle analysis was reduced markedly. Therefore, in cervical cancer, cytokeratin labelling can optimize both the determination of DNA ploidy and cell-cycle analysis.