keratoconus ( kc ) is a bilateral noninflammatory disease of the cornea characterized by progressive corneal thinning and ectasia . introduced in 2003 , corneal crosslinking ( cxl ) was the first treatment aimed at the pathogenetic cause of kc by potentially changing the intrinsic biomechanical properties of the corneal collagen . histologically , cxl causes an increase of the fiber diameter and chemical bonding between corneal microstructural components , leading to a higher mechanical stiffness of the cornea [ 3 , 4 ] . a reduced susceptibility to enzymatic degradations has also been described [ 5 , 6 ] . over the past few years , long - term studies concentrating on in vivo topography and tomographic analyses have been published to demonstrate that cxl is an effective treatment for stopping the natural course of progressive kc [ 79 ] . the next step in extending our knowledge of cxl , and thereby in optimizing treatment , is to gain a better understanding of not only the morphological but also the in vivo biomechanical effects of cxl . we could then make a better evaluation of the different cxl strategies , which may possibly enable individualized therapy strategies . the aim of the current study was to analyze biomechanical changes of the cornea after cxl for progressive kc by applying in vivo corneal visualization scheimpflug technology ( cst ) combined with new research software ( version 6.07r08 ) . this retrospective study was performed in cooperation between the department of ophthalmology , university medical center hamburg - eppendorf , and the care vision eye clinic hamburg . inclusion criteria for cxl werediagnosis of a progressive keratoconus that is pentacam topography documented progression of maximum k - values > 0.5  d within 6 months . the diagnosis of kc was confirmed by asymmetry of the corneal surface ( kisa% ) of > 100 . further , all patients were classified as stage 1 or 2 according to the amsler - krumeich classification ; best corrected visual accuracy of at least 0.4 logmar;absence of corneal scars;signed consent form including the information of the off - label status of the therapy . diagnosis of a progressive keratoconus that is pentacam topography documented progression of maximum k - values > 0.5  d within 6 months . the diagnosis of kc was confirmed by asymmetry of the corneal surface ( kisa% ) of > 100 . further , all patients were classified as stage 1 or 2 according to the amsler - krumeich classification ; best corrected visual accuracy of at least 0.4 logmar ; absence of corneal scars ; signed consent form including the information of the off - label status of the therapy . exclusion criteria for cxl werecentral corneal thickness < 400 microns ( even after intraoperative corneal swelling with a hypotonic solution);pregnancy;severe dry eye syndrome;inflammation of the ocular surface , the anterior chamber or the eyelids . central corneal thickness < 400 microns ( even after intraoperative corneal swelling with a hypotonic solution ) ; severe dry eye syndrome ; inflammation of the ocular surface , the anterior chamber or the eyelids . biomechanical analyses were performed with the corvis st ( cst ; oculus inc . , dudenhofen , germany ) . after using the automated export function of the cst , the data were recalculated by applying the cst research software version 6.07r08 developed by oculus . moreover , it provides extra quality scores that help in further assessing the value of the data . the new research software adds the following parameters.deflection amplitude [ mm ] ( figure 1 ) : displacement of the corneal apex in reference to the overlaid cornea in initial state ( blue line ) . it is the sum of deflection amplitude and whole eye movement ( green line).deflection length [ mm ] ( figure 2 ) : length of a line ( blue line , figure 2 ) that describes the deflected part of the cornea compared to the undeformed cornea in the initial state ( red dotted line ) . the two end points ( blue circles ) are fitted to the positions where the shape of the outskirts of the cornea does not differ from the cornea in the initial state . this allows more robust information to be obtained on the applanated part of the cornea at the time of the first and second applanation compared to just the applanation length.radius ( 3p ) [ mm ] : radius of curvature at maximum deformation , 3-point - fit.a1 deflection length [ mm ] : deflection length at the time of the first applanation.hc deflection length [ mm ] : deflection length at the time of the highest concavity.a2 deflection length [ mm ] : deflection length at the time of the second applanation.hc deflection amp . deflection amplitude [ mm ] ( figure 1 ) : displacement of the corneal apex in reference to the overlaid cornea in initial state ( blue line ) . it is the sum of deflection amplitude and whole eye movement ( green line ) . deflection length [ mm ] ( figure 2 ) : length of a line ( blue line , figure 2 ) that describes the deflected part of the cornea compared to the undeformed cornea in the initial state ( red dotted line ) . the two end points ( blue circles ) are fitted to the positions where the shape of the outskirts of the cornea does not differ from the cornea in the initial state . this allows more robust information to be obtained on the applanated part of the cornea at the time of the first and second applanation compared to just the applanation length . radius ( 3p ) [ mm ] : radius of curvature at maximum deformation , 3-point - fit . a1 deflection length [ mm ] : deflection length at the time of the first applanation . hc deflection length [ mm ] : deflection length at the time of the highest concavity . a2 deflection length [ mm ] : deflection length at the time of the second applanation . the cst analyses were performed with a median of 0 days before and 84 days ( 3 month ) after cxl . the cst displays a quality specification grade ( qs ) based on the patient 's alignment and the integrity of the data record for every analysis . only cst analyses with a status  ok  for all available qs were included in the statistical analyses . if the patient had used contact lenses , a minimum of 14 days ( hard lenses ) or 4 days ( soft lenses ) of contact lens abstinence was maintained . to avoid a potential bias attributed to diurnal variations of the corneal thickness and the anterior and posterior corneal surface , both the pre - cxl and the 3-month post - cxl analyses oxybuprocaine 0.4% eye drops were applied for preoperative local anesthesia . after inserting the eyelid speculum , the central 9  mm of the corneal epithelium was cautiously removed using 20% alcohol solution ( 20 seconds ) and a blunt knife . after de - epithelialization , an ultrasound - pachymetry was performed to ensure a central corneal thickness > 400  m . in case of a central corneal thickness > 400  m , a photosensitizer , riboflavin 0.1% solution ( 10  mg riboflavin-5-phosphate in 10  ml dextran - t-500 20% solution ) was applied every 2 minutes for 30 minutes . if the pachymetry was < 400  m , we applied 0.1% hypo - osmolar riboflavin solution . after 30 minutes , the application of riboflavin was followed by another pachymetry to confirm a central corneal thickness of at least 400  m . if the corneal thickness was thinner then 400  m , we continued the application of the hypotonic solution until the pachymetry exceeded 400  m and continued with the hypotonic solution throughout the irradiation . if the central pachymetry was > 400  m in both measurements , we applied the riboflavin 0.1% solution as described above . the uva irradiation was started using uv diodes ( 370  nm ; peschke lasertechnik , waldshut - tiengen , germany ) with the uva light focusing on the cornea while protecting the limbus . before each treatment , the desired irradiance of 3  mw / cm was controlled with a uva meter ( lasermate - q ; laser 2000 , wessling , germany ) and , if necessary , regulated with the potentiometer . the patient 's cornea was irradiated with the uva - light diodes for 30 minutes . treated eyes were dressed with a soft contact lens bandage for 4 days and were medicated with antibiotics ( ofloxacin drops 4 times / day ) , nonsteroidal anti - inflammatory drugs ( diclofenac drops 4 times / day ) , and lubricants ( phospholipidic microemulsion drops tapered 8 times / day ) . after cl removal on 4th postoperative day the therapeutic regimen was changed to fluorometholone 0.2% drops ( 4 times / day ) and lubricants ( phospholipidic microemulsion drops , 8 times / day ) . during the 5th and 10th week after cxl , fluorometholone was reduced by 1 eye drop every two weeks . informed consent for retrospective data analysis and approval of the institutional review board ( irb)/ethics committee for the study were obtained . before statistical analyses , all cst parameters were automatically exported into a spreadsheet program ( microsoft office excel ) using the original software of the device . the data were recalculated using the new research software of the cst developed by oculus ( v.6.07r08 ) . for statistical analyses , the general purpose statistical software ( stata version 11.0 ; statacorp ) was applied . for normal distributions , a t - test was used to test for statistically significant differences between pre- and postoperative analyses . if a parameter was not normally distributed , a wilcoxon matched - pairs test was used . our database contained records of 22 patients with progressive kc who received cst analyses before and after cxl . of these , 17 eyes of 17 patients ( 2 females , 15 males ; mean age 27 years ; min / max : 20/47 years ) displayed cst analyses with high quality results before and median 3 months after cxl . more than 75% of the examinations were conducted only minutes before the treatment and mean followup was 3 months . high values of the belin ambroso index ( bad_d ) , the topographic astigmatism and/or the maximum keratometry of the corneal surface ( kmax ) could be demonstrated for every patient before the treatment . table 2 displays the changes of the exported cst parameters before and 3 months after cxl . of the 24 automatically exported parameters of the cst , 4 parameters displayed statistically significant differences between pre- and postoperative examination : the intraocular pressure ( iop ; median : + 3  mmhg ) , the central corneal pachymetry ( pachy ; mean : 35  m ) , the timespan between the air impulse release and the first applanation of the cornea ( a1time ; median : + 0.12  ms ) , and the timespan between the air impulse release and the second applanation of the cornea , which occurs when the cornea passed the point of maximum impression and is on its way back to the initial state ( a2time ; median : 37  ms ) ( see also table 3 ) . all these statistically significant changes indicate an increase of the corneal stiffness after the treatment . even considering the parameters without statistically significant changes , none of the analyzed parameters demonstrated an alteration , which might suggest a worsening of the corneal biomechanical properties after cxl . we calculated the sample sizes for the new deflection parameters based on our data ( table 4 ) . based on the distribution and the number of subjects included ( see also n in table 2 ) the required sample sizes vary between 21 and > 10,000 . it should be born in mind that the calculated sample size also strongly depends on the follow - up period . we identified statistically significant differences between four parameters obtained before and 3 months after cxl : iop , central corneal pachymetry , a1time ,  and  a2time .  over the past few years , several methods for measuring the geometric structure of the cornea have been used for analyzing corneal geometrics and to describe corneal pathologies . these devices have improved our understanding of corneal pathologies and helped us to identify pathologies like kc in its early stages . besides these improvements in structural analyses , in vivo analyses of biomechanical changes have been developed . the first device allowing in vivo analyses of the cornea was the ocular response analyzer ( ora ) ( reichert technologies ) [ 15 , 16 ] . only a few study groups have analyzed in vivo biomechanical changes in progressive kc after cxl [ 1720 ] . unfortunately , ora analyses of post - cxl changes have not yielded consistent findings [ 17 , 18 , 20 , 21 ] . recently , oculus introduced a new device for in vivo analyses of corneal biomechanics called  corneal visualization scheimpflug technology  ( cst ) . like the ora , it uses a precise collimated air pulse to cause the cornea to move inwards . however , in contrast to the ora , it uses high - speed scheimpflug technology to follow the movement of the cornea throughout the whole dynamic process of inward and outwards motion . in this way a range of parameters are generated which enable complex analyses of the viscoelastic properties of the cornea . until now , only a few studies comparing biomechanical properties of kc and normal eyes have been published [ 13 , 22 ] . tian et al . , using the cst software version 1.00r30 , found the deflection amplitude ( da ) to be the best parameter for differentiating between kc and normal eyes ( ne ) by demonstrating a higher da in kc ( their  da  is equivalent with our  defampmax ( mm )  ) . they also demonstrated a lower concavity curvature and faster corneal applanation velocity in kc compared to ne . , comparing data of 45 kc eyes and 103 ne , also found the da to be the potentially strongest parameter for differentiating between kc and ne , with da being higher in kc ( kc : 1.37  0.21  mm ; ne : 1.05  0.11  mm , p < 0.001 ) . however , because of the only minor roc ( receiver operating characteristic ) areas with no ideal cut - off values , they concluded that the da may be a useful adjunct in kc assessment and monitoring but can not solely discriminate between normal and keratoconic corneas . used the cst for analyzing biomechanical changes of the cornea after cxl for progressive kc . they compared the results of accelerated versus conventional cxl in progressive kc by applying ora and cst with a follow - up of 1 year . similar to our study , they included subjects with kc classified as first or second stage according to the amlser - krumeich classification . unfortunately , they did not give information about the criteria of kc progression leading to the indication for cxl . as in previous studies , they could not demonstrate statistically significant changes after conventional cxl using the ora for in vivo biomechanical analyses . however , using cst analyses , they found changes in three parameters : the deformation amplitude ( da ; 0.02  mm ) , the distance between corneal bending points (  peak distance  ; + 0.42  mm ) and the radius of the curvature at the time of highest concavity of the cornea ( + 0.1  mm ) . although these changes indicate a higher stiffness of the cornea , the changes in these parameters were not statistically significant at 1 year after conventional cxl . we demonstrated statistically significant changes in the a1time ( median : + 0.12  ms ) and the a2time ( median : 0.37  ms ) . in addition , the iop ( median : + 3  mmhg ) and the central corneal pachymetry ( pachy ; mean : 35  m ) demonstrated statistically significant changes . in line with previously published data , the da decreased slightly , but not on a statistically significant level ( 0.9  mm , p = 0.155 ) . also the peak distance and the radius at the time of the highest concavity changed with the same direction as that demonstrated by tomami et al . , but again did not reach a statically significant level ( peak distance : 0.49  mm , p = 0.165 ; radius : + 0.17 mm ; p = 0.488 ) . indicate an increase in the corneal resistance after the treatment . because of the increased stiffness  of the corneal tissue , the time until the cornea reaches the status of the first applanation ( a1time ) increased and the time of the second applanation ( after the cornea passed the point of maximum deformation [ a2time ] ) decreased . correspondingly , tian et al . demonstrated a statistically significant longer  a1time  and a shorter  a2time  in ne than in kc on analyzing the biomechanical properties of the cornea in kc and ne with the cst . showed that the biomechanical analyses measured with the cst are strongly influenced by the iop and the pachymetry . this is of curial importance , especially in cross - sectional - analyses . in longitudinal studies , the iop or the pachymetry should not matched or accounted before statistical analyses to avoid generating confounding factors . an increase in pachymetry leads to an increase in the measured iop values since more pressure is needed to applanate the cornea regardless of the real iop [ 24 , 25 ] . we demonstrated a statistically significant increase in the iop despite a decrease of the pachymetry . this relationship can be seen as a further biomechanical indication of an increase of corneal stiffness . accordingly , tian et al . , analyzing kc and ne , demonstrated a statistically significantly lower iop in their kc group . the initial decrease of corneal pachymetry after cxl has been described in several studies [ 2628 ] . because pre- and post - cxl measurements were performed between 8 and 10 a.m. , the diurnal change of iop should be negligible [ 29 , 30 ] . several study groups have already demonstrated a high repeatability and accuracy of iop and pachymetry measurement with the cst [ 23 , 31 , 32 ] . focusing on deformation - related parameters , as in the mentioned above , the sum of the deflection ( real corneal apex movement ) and the whole eye movement are analyzed . as shown in the methods section , the deflection describes the displacement of the corneal apex in reference to the overlaid cornea in its initial state . therefore , the movement of the corneal apex is compensated by the whole eye movement and only the  real  movement of the cornea is described . unfortunately , despite applying the new test software , we could not demonstrate further statistically significant differences in the deflection - based parameters . this is probably related to the small sample size and the short follow - up of our study . different study groups have demonstrated ongoing changes of the cornea even years after the cxl [ 79 ] . the strength of our study is the methodological standardization : we only implemented cst analyses with objectively measured high - quality standards ( included qs  ok  or model deviation  ; excluding all analyses with an  alignment  or  lost images  warning ) . excluding confounding factors such as wrong alignment is crucial because the cst measures corneal changes caused by a precise collimated air pulse . in addition , 76% of the included pre - cxl analyses were performed only minutes before the cxl , reducing the probability of potential changes between the last examination before the cxl and the time of the treatment . further , all analyses were performed between 8 and 10 a.m. to reduce diurnal variations . as already mentioned , besides an improved feedback regarding the quality of the analyses ( additional qs scores ) , this version provides ( deflection - based ) parameters that are not compromised by back and forth movements of the eye that occur as a reaction to the air impulse during the measurement . additionally , 24% of the pre - cxl analyses were not performed immediately before cxl but within 6 weeks before the treatment ( range 1644 days before cxl ) . this might affect the results due to potentially unknown biomechanical changes between the pre - cxl measurement and the treatment . therefore , a prospective study design with requested cst on the same day as cxl would be helpful to minimize the time interval between analysis and treatment . alike , the time range of the post - cxl examinations might bias the results . as displayed in table 1 , 89% of the examinations post - cxl were conducted 7090 days after the treatment . these outliers potentially affect the results by either not detecting subtle changes due to a too short timespan after cxl , or overrating changes in the context of our median 3 month follow up . unfortunately , we could not find other prospective or retrospective studies analyzing changes after cxl for progressive kc which report the time range of their follow up intervals pre- and post cxl . because of a high percentage of examinations conducted only few minutes before the treatment ( 76% of the included examinations ) and almost 90% of the examinations performed within a time range of 20 days at the 3 month follow up , we think that our results contribute to the understanding of biomechanical changes after cxl for progressive kc . we identified two parameters , a1time ' and a2time , that indicate an improvement of the corneal biomechanical properties after corneal cxl for progressive kc . none of the other cst parameters revealed statistical significant changes , demonstrating at least a stabilizing effect of cxl on corneal biomechanics .
purpose . to evaluate the effect of corneal crosslinking in progressive keratoconus by applying in vivo corneal visualization scheimpflug technology 
 . design . 
 longitudinal retrospective study . subjects and controls . 
 seventeen eyes of patients treated with corneal crosslinking for progressive keratoconus 
 . methods . 
 corneal visualization scheimpflug technology analyses ( research software version 6.07r08 ) of subjects with progressive keratoconus before and 3 months after corneal crosslinking ( cxl ) were reviewed retrospectively . 
 t - test ( for normal distribution ) and wilcoxon matched - pairs test ( if not normally distributed ) were used to test for statistically significant differences between pre- and post - cxl analyses . results . 
 we demonstrated statistically significant differences for the intraocular pressure ( median : + 3  mmhg , p = 0.004 ) , the central corneal pachymetry ( pachy ; mean : 35  m , p < 0.001 ) , the timespan between the air impulse release and the first applanation of the cornea ( a1time ; median : + 0.12  ms , p < 0.05 ) , and the timespan between the air impulse release and the second applanation of the cornea ( a2time ; median : 37  ms , p < 0.05 ) . conclusions . with the a1time and the a2time , we identified two parameters that demonstrated a statistically significant improvement of the biomechanical properties of the cornea after cxl . despite the known initial decrease of the pachymetry after cxl , none of the analyzed parameters indicated a progression of the keratoconus .