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Purpose: High-resolution images of glaucomatous damage to the retinal nerve fiber layer (RNFL) were obtained with an adaptive optics-scanning light ophthalmoscope (AO-SLO) and used as a basis for comparisons between en face slab images and thickness maps derived from optical coherence tomography (OCT) scans., Methods: Wide-field (9 × 12 mm) cube scans were obtained with swept-source OCT (DRI-OCT) from six eyes of six patients. All eyes had a deep defect near fixation as seen on a 10-2 visual field test. Optical coherence tomography en face images, based on the average reflectance intensity, were generated (ATL 3D-Suite) from 52-μm slabs just below the vitreal border of the inner limiting membrane. The RNFL thickness maps were generated from the same OCT data. Both were compared with the AO-SLO peripapillary images that were previously obtained., Results: On AO-SLO images, three eyes showed small regions of preserved and/or missing RNFL bundles within the affected region. Details in these regions were seen on the OCT en face images but not on the RNFL thickness maps. In addition, in the healthier hemi-retinas of two eyes, there were darker, arcuate-shaped regions on en face images that corresponded to abnormalities seen on AO-SLO. These were not seen on RNFL thickness maps., Conclusions: Details of local glaucomatous damage, missing or easily overlooked on traditional OCT RNFL thickness analysis used in clinical OCT reports, were seen on OCT en face images based on the average reflectance intensity. While more work is needed, it is likely that en face slab imaging has a role in the clinical management of glaucoma.

Purpose: To relate changes in retinal function and retinal nerve fiber layer (RNFL) retardance to loss of RNFL thickness and optic nerve axon counts in a nonhuman primate (NHP) model of experimental glaucoma (EG)., Methods: Bilateral longitudinal measurements of peripapillary RNFL thickness (spectral-domain optical coherence tomography, SDOCT; Spectralis), retardance (GDxVCC), and multifocal electroretinography (mfERG; VERIS) were performed in 39 NHP at baseline (BL; median, 5 recordings; range, 3-10) and weekly after induction of unilateral EG by laser photocoagulation of the trabecular meshwork. Multifocal ERG responses were high-pass filtered (>75 Hz) to measure high- and low-frequency component (HFC and LFC) amplitudes, including LFC features N1, P1, and N2. High-frequency component amplitudes are known to specifically reflect retinal ganglion cell (RGC) function. Complete (100%) axon counts of orbital optic nerves were obtained in 31/39 NHP., Results: Postlaser follow-up was 10.4 ± 7.9 months; mean and peak IOP were 18 ± 5 and 41 ± 11 mm Hg in EG eyes, 11 ± 2 and 18 ± 6 mm Hg in control (CTL) eyes. At the final available time point, RNFL thickness had decreased from BL by 14 ± 14%, retardance by 20 ± 11%, and the mfERG HFC by 30 ± 17% (P < 0.0001 each). Longitudinal changes in retardance and HFC were linearly related to RNFL thickness change (R2 = 0.51, P < 0.0001 and R2 = 0.22, P = 0.002, respectively); LFC N2 was weakly related but N1 or P2 (N1: R2 = 0.07, P = 0.11; P1: R2 = 0.04, P = 0.24; N2: R2 = 0.13, P = 0.02). At zero change from BL for RNFL thickness (Y-intercept), retardance was reduced by 11% (95% confidence interval [CI]: -15.3% to -6.8%) and HFC by 21.5% (95% CI: -28.7% to -14.3%). Relative loss of RNFL thickness, retardance, and HFC (EG:CTL) were each related to axon loss (R2 = 0.66, P < 0.0001; R2 = 0.42, P < 0.0001; R2 = 0.42, P < 0.0001, respectively), but only retardance and HFC were significantly reduced at zero relative axon loss (Y-intercept; retardance: -9.4%, 95% CI: -15.5% to -3.4%; HFC: -10.9%, 95% CI: -18.6% to -3.2%; RNFL thickness: +1.8%, 95% CI: -4.9% to +5.4%)., Conclusions: Retinal nerve fiber layer retardance and RGC function exhibit progressive loss from baseline before any loss of RNFL thickness or orbital optic nerve axons occurs in NHP EG. These in vivo measures might serve as potential biomarkers of early-stage glaucomatous damage preceding axon loss and RGC death.

Purpose: To introduce quantitative postmortem lamina cribrosa (LC) microarchitecture (LMA) assessment and characterize beam diameter (BD), pore diameter (PD), and connective tissue volume fraction (CTVF) in 21 normal monkey eyes., Methods: Optic nerve heads (ONHs) underwent digital three-dimensional (3D) reconstruction and LC beam segmentation. Each beam and pore voxel was assigned a diameter based on the largest sphere that contained it before transformation to one of twelve 30° sectors in a common cylinder. Mean BD, PD, and CTVF within 12 central and 12 peripheral subsectors and within inner, middle, and outer LC depths were assessed for sector, subsector, and depth effects by analysis of variance using general estimating equations. Eye-specific LMA discordance (the pattern of lowest connective tissue density) was plotted for each parameter., Results: The ranges of mean BD, PD, and CTVF were 14.0 to 23.1 μm, 20.0 to 35.6 μm, and 0.247 to 0.638, respectively. Sector, subsector, and depth effects were significant (P < 0.01) for all parameters except subsector on CTVF. Beam diameter and CTVF were smaller and PD was larger within the superior-temporal (ST) and inferior-temporal (IT) sectors (P < 0.05). These differences were enhanced within the central versus peripheral subsectors. Beam diameter and CTVF were larger and PD was smaller (P < 0.05) within the middle LC layer. Lamina cribrosa microarchitecture discordance most commonly occurred within the ST and IT sectors, varied by eye, and generally diminished as CTVF increased., Conclusions: Our data support previous characterizations of diminished connective tissue density within the ST and IT ONH regions. The clinical importance of eye-specific LMA discordance warrants further study., (Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: To characterize age-related differences in the magnitude of spectral-domain optical coherence tomography (SD-OCT) structural change in early experimental glaucoma (EG)., Methods: Both eyes from four young (1.4-2.6 years) and four old (18.6-21.9 years) rhesus monkeys were imaged at least three times at baseline, and then every 2 weeks after laser-induced, chronic, unilateral IOP elevation until the onset of EG (confocal scanning laser tomographic surface change confirmed twice). Two to 20 weeks after EG onset, animals were euthanized and optic nerve axon counts for all eyes were performed. Masked operators delineated retinal and ONH landmarks in 40 radial B-scans from each eye and imaging session to quantify change from baseline in five SD-OCT neural and connective tissue parameters. The effects of EG, age, and EG × age interactions on the magnitude, rate (magnitude per postlaser time), and IOP responsiveness (magnitude per cumulative IOP insult) of postlaser parameter change were individually assessed using general estimating equation models., Results: Presac SD-OCT RNFLT and minimum rim width change and postmortem axon loss was not significantly different in old compared with young EG eyes. The rate of change and IOP responsiveness of the parameters anterior lamina cribrosa surface depth relative to Bruch's membrane opening (BMO) and BMO depth relative to peripheral Bruch's membrane were significantly lower (P < 0.05) in the old compared with the young EG eyes., Conclusions: At similar postlaser times, levels of cumulative IOP insult and axonal damage, SD-OCT-detected ONH connective tissue structural change is greater in young compared with old monkey EG eyes., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: To evaluate the effect of age on optic nerve axon counts, spectral-domain optical coherence tomography (SDOCT) scan quality, and peripapillary retinal nerve fiber layer thickness (RNFLT) measurements in healthy monkey eyes., Methods: In total, 83 healthy rhesus monkeys were included in this study (age range: 1.2-26.7 years). Peripapillary RNFLT was measured by SDOCT. An automated algorithm was used to count 100% of the axons and measure their cross-sectional area in postmortem optic nerve tissue samples ( N = 46). Simulation experiments were done to determine the effects of optical changes on measurements of RNFLT. An objective, fully-automated method was used to measure the diameter of the major blood vessel profiles within each SDOCT B-scan., Results: Peripapillary RNFLT was negatively correlated with age in cross-sectional analysis ( P < 0.01). The best-fitting linear model was RNFLT (μm) = -0.40 × age (years) + 104.5 μm ( R 2 = 0.1, P < 0.01). Age had very little influence on optic nerve axon count; the result of the best-fit linear model was axon count = -1364 × Age (years) + 1,210,284 ( R 2 < 0.01, P = 0.74). Older eyes lost the smallest diameter axons and/or axons had an increased diameter in the optic nerve of older animals. There was an inverse correlation between age and SDOCT scan quality ( R = -0.65, P < 0.0001). Simulation experiments revealed that approximately 17% of the apparent cross-sectional rate of RNFLT loss is due to reduced scan quality associated with optical changes of the aging eye. Another 12% was due to thinning of the major blood vessels., Conclusions: RNFLT declines by 4 μm per decade in healthy rhesus monkey eyes. This rate is approximately three times faster than loss of optic nerve axons. Approximately one-half of this difference is explained by optical degradation of the aging eye reducing SDOCT scan quality and thinning of the major blood vessels., Translational Relevance: Current models used to predict retinal ganglion cell losses should be reconsidered.

Purpose: To quantify the effects of using the fovea to Bruch's membrane opening (FoBMO) axis as the nasal-temporal midline for 30° sectoral (clock-hour) spectral domain optical coherence tomography (SDOCT) optic nerve head (ONH) minimum rim width (MRW) and area (MRA) calculations., Methods: The internal limiting membrane and BMO were delineated within 24 radial ONH B-scans in 222 eyes of 222 participants with ocular hypertension and glaucoma. For each eye the fovea was marked within the infrared reflectance image, the FoBMO angle (θ) relative to the acquired image frame (AIF) horizontal was calculated, the ONH was divided into 30° sectors using a FoBMO or AIF nasal/temporal axis, and SDOCT MRW and MRA were quantified within each FoBMO vs. AIF sector. For each sector, focal rim loss was calculated as the MRW and MRA gradients (i.e. the difference between the value for that sector and the one clockwise to it divided by 30°). Sectoral FoBMO vs. AIF discordance was calculated as the difference between the FoBMO and AIF values for each sector. Generalized estimating equations were used to predict the eyes and sectors of maximum FoBMO vs. AIF discordance., Results: The mean FoBMO angle was -6.6±4.2° (range: -17° to +7°). FoBMO vs. AIF discordance in sectoral mean MRW and MRA was significant for 7 of 12 and 6 of 12 sectors, respectively (p<0.05, Wilcoxon test, Bonferroni correction). Eye-specific, FoBMO vs. AIF sectoral discordance was predicted by sectoral rim gradient (p<0.001) and FoBMO angle (p<0.001) and achieved maximum values of 83% for MRW and 101% for MRA., Conclusions: Using the FoBMO axis as the nasal-temporal axis to regionalize the ONH rather than a line parallel to the AIF horizontal axis significantly influences clock-hour SDOCT rim values. This effect is greatest in eyes with large FoBMO angles and sectors with focal rim loss.

Purpose: To test the hypothesis that optic nerve head (ONH) deformation manifesting as changes in its mean surface height precedes thinning of the peripapillary retinal nerve fiber layer (RNFL) in experimental glaucoma (EG)., Methods: 68 rhesus macaque monkeys each had three or more baseline imaging sessions under manometric intraocular pressure (IOP) control to obtain average RNFL thickness (RNFLT) and the ONH surface topography parameter mean position of the disc (MPD). Laser photocoagulation was then applied to the trabecular meshwork of one eye to induce chronic, mild-to-moderate IOP elevation and bi-weekly imaging continued. Event analysis was applied to determine for each parameter when an 'endpoint' occurred (signficant change from baseline) for eight different endpoint criteria. Specificity was assessed in the group of 68 fellow control eyes. Classical signal detection theory and survival analysis were used to compare MPD with RNFLT., Results: Regardless of the endpoint criterion, endpoints were always more frequent for MPD than for RNFLT. The discriminability index (d') was 2.7 ± 0.2 for MPD and 1.9 ± 0.2 for RNFLT (p<0.0001). Endpoints were reached by MPD an average of 1-2 months earlier than by RNFLT (p<0.01). At the onset of the first specific, detectable MPD change in EG eyes, there was still no significant change in RNFLT on average (p=0.29) and only 25% of individual eyes exhibited signficant reduction. In contrast, at onset of signficant RNFLT change, MPD had already changed an average of 101 µm from baseline (p<0.0001) and 71% of the individual eyes had exhibited significant change. The magnitude of MPD change was more than could be explained on the basis of axon loss alone., Conclusions: This study demonstrates that the average surface height of the ONH changes prior to any detectable loss of average peripapillary RNFL thickness in non-human primate eyes with experimental glaucoma.

Purpose: Longitudinal measurements of peripapillary RNFL thickness and retardance were compared in terms of time to reach onset of damage and time to reach a specific progression endpoint., Methods: A total of 41 rhesus macaques with unilateral experimental glaucoma (EG) each had three or more weekly baseline measurements in both eyes of peripapillary RNFL thickness (RNFLT) and retardance. Laser photocoagulation was then applied to the trabecular meshwork of one eye to induce chronic elevation of intraocular pressure and weekly imaging continued. Pairwise differences between baseline observations were sampled by bootstrapping to determine the 95% confidence limits of each measurement's repeatability. The first two sequential measurements below the lower confidence limit defined the endpoint for each parameter. Segmented linear and exponential decay functions were fit to each RNFL-versus-time series to determine the time to damage onset., Results: In all, 29 (71%) of the EG eyes reached endpoint by RNFL retardance and 25 (61%) reached endpoint by RNFLT. In total, 33 (80%) reached endpoint by at least one of the RNFL parameters and 21 (51%) reached endpoint by both RNFL parameters. Of the 33 EG eyes reaching any endpoint, a larger proportion reached endpoint first by retardance (n = 26, 79%) than did by RNFLT (n = 7, 21%; P = 0.002). Survival analysis indicated a shorter time to reach endpoint by retardance than by RNFLT (P < 0.001). Of the 21 EG eyes that reached endpoint by both measures, the median duration to endpoint was 120 days for retardance and 223 days for RNFLT (P = 0.003, Wilcoxon test). The time to onset was faster for retardance than that for RNFLT based on either segmented fits (by 31 days; P = 0.008, average R(2) = 0.89) or exponential fits (by 102 days; P = 0.01, average R(2) = 0.89)., Conclusions: The onset of progressive loss of RNFL retardance occurs earlier than the onset of RNFL thinning. Endpoints of progressive loss from baseline also occurred more frequently and earlier for RNFL retardance as compared with RNFLT.

Purpose: We determined the relationship between total optic nerve axon counts and peripapillary retinal nerve fiber layer thickness (RNFLT) measured in vivo by spectral domain optical coherence tomography (SDOCT)., Methods: A total of 22 rhesus macaques had three or more baseline measurements in both eyes of peripapillary RNFLT made by SDOCT. Laser photocoagulation then was applied to the trabecular meshwork of one eye to induce chronic unilateral IOP elevation. SDOCT measurements of RNFLT continued approximately every two weeks until the predefined study endpoint was reached in each animal. At endpoint, animals were sacrificed and the optic nerve was sampled approximately 2 mm behind the globe to obtain thin sections for histologic processing and automated axon counting across 100% of the optic nerve cross-sectional area., Results: At the final imaging session, the average loss of RNFLT was 20 ± 21%, ranging from essentially no loss to nearly 65% loss. Total optic nerve axon count in control eyes ranged from 812,478 to 1,280,474. The absolute number of optic nerve axons was related linearly to RNFLT (axon count = 12,336 × RNFLT((μm)) - 257,050, R(2) = 0.65, P < 0.0001), with a Pearson correlation coefficient of 0.81. There also was a strong linear relationship between relative optic nerve axon loss (glaucomatous-to-control eye) and relative RNFLT at the final imaging session, with a slope close to unity but a significantly negative intercept (relative axon loss((%)) = 1.05 × relative RNFLT loss((%)) - 14.4%, R(2) = 0.75, P < 0.0001). The negative intercept was robust to variations of fitted model because relative axon loss was -14% on average for all experimental glaucoma (EG) eyes within 6% (measurement noise) of zero relative loss., Conclusions: There is a strong linear relationship between total optic nerve axon count and RNFLT measured in vivo by SDOCT. However, substantial loss of optic nerve axons (∼10%-15%) exists before any loss of RNFLT manifests and this discrepancy persists systematically throughout a wide range of damage.

Purpose: To compare peripapillary retinal nerve fiber layer thickness (RNFLT), RNFL retardance, and retinal function at the onset of optic nerve head (ONH) surface topography change in experimental glaucoma (EG)., Methods: Thirty-three rhesus macaques had three or more weekly baseline measurements in both eyes of ONH surface topography, peripapillary RNFLT, RNFL retardance, and multifocal electroretinography (mfERG). Laser photocoagulation was then applied to the trabecular meshwork of one eye to induce chronic elevation of IOP and weekly recordings continued alternating between ONH surface topography and RNFLT during one week and RNFL retardance and mfERG the next week. Data were pooled for the group at the onset of ONH surface topography change in each EG eye, which was defined as the first date when either the mean position of the disc (MPD) fell below the 95% confidence limit of each eye's individual baseline range and/or when the topographic change analysis (TCA) map was subjectively judged as having demonstrated change, whichever came first. Analysis of variance with post hoc tests corrected for multiple comparisons were used to assess parameter changes., Results: At onset of ONH surface topography change, there was no significant difference for RNFLT versus baseline or fellow control eyes. RNFL retardance and mfERG were significantly reduced in the recordings just prior (median of 9 days) to ONH onset (P < 0.01) and had progressed significantly (P < 0.001) an average of 17 days later (median of 7 days after ONH onset). RNFLT did not exhibit significant thinning until 15 days after onset of ONH surface topography change (P < 0.001)., Conclusions: These results support the hypothesis that during the course of glaucomatous neurodegeneration, axonal cytoskeletal and retinal ganglion cell functional abnormalities exist before thinning of peripapillary RNFL axon bundles begins.

Purpose: To describe an algorithm and software application (APP) for 100% optic nerve axon counting and to compare its performance with a semi-automated manual (SAM) method in optic nerve cross-section images (images) from normal and experimental glaucoma (EG) nonhuman primate (NHP) eyes., Methods: ON cross sections from eight EG eyes from eight NHPs, five EG and five normal eyes from five NHPs, and 12 normal eyes from 12 NHPs were imaged at 100×. Calibration (n = 500) and validation (n = 50) image sets ranging from normal to end-stage damage were assembled. Correlation between APP and SAM axon counts was assessed by Deming regression within the calibration set and a compensation formula was generated to account for the subtle, systematic differences. Then, compensated APP counts for each validation image were compared with the mean and 95% confidence interval of five SAM counts of the validation set performed by a single observer., Results: Calibration set APP counts linearly correlated to SAM counts (APP = 10.77 + 1.03 [SAM]; R(2) = 0.94, P < 0.0001) in normal to end-stage damage images. In the validation set, compensated APP counts fell within the 95% confidence interval of the SAM counts in 42 of the 50 images and were within 12 axons of the confidence intervals in six of the eight remaining images. Uncompensated axon density maps for the normal and EG eyes of a representative NHP were generated., Conclusions: An APP for 100% ON axon counts has been calibrated and validated relative to SAM counts in normal and EG NHP eyes.

PURPOSE. To evaluate the effect of acutely elevated intraocular pressure (IOP) on retinal thickness and optic nerve head (ONH) structure in the rat eye by spectral domain-optical coherence tomography (SD-OCT). METHODS. Fourteen adult male Brown-Norway rats were studied under anesthesia (ketamine/xylazine/acepromazine, 55:5:1 mg/kg intramuscularly). Both eyes were imaged by SD-OCT on two baseline occasions several weeks before and again 2 and 4 weeks after the acute IOP imaging session. During the acute IOP session, SD-OCT imaging was performed 10 minutes after IOP was manometrically set at 15 mm Hg and then at 10, 30, and 60 minutes after IOP had been elevated to 50 mm Hg (n = 8) and again 10 and 30 minutes after IOP had been lowered back to 15 mm Hg (recovery). In two additional groups, IOP elevation was set to 70 mm Hg (n = 4) or 40 mm Hg (n = 2). Acute IOP results are reported for a pattern of 49 horizontal B-scans spanning a 20° square and follow-up results for peripapillary circular B-scans. Retinal and retinal nerve fiber layer (RNFL) thicknesses were measured with custom software by manual image segmentation. Friedman and Dunn's tests were used to assess acute and longer-term effects of acute IOP elevation. RESULTS. Acute IOP elevation to 50 mm Hg caused rapid (within seconds) deformation of the ONH and peripapillary structures, including posterior displacement of the ONH surface and outward bowing of peripapillary tissue; retinal thickness decreased progressively from 10 to 30 to 60 minutes by 16%, 18%, and 20% within the area of Bruch's membrane opening (BMO; P < 0.0001) by 8%, 9%, and 11% within the central 10° (excluding the BMO; P < 0.0001) but only by 1%, 2%, and 2.4% beyond the central 10° (P < 0.0001). Recovery was progressive and nearly complete by 30 minutes. Acute IOP elevation to 40 and 70 mm Hg produced similar structural changes, but 70 mm Hg also interfered with retinal blood flow. There were no changes in peripapillary retinal or RNFL thickness (P = 0.08 and P = 0.16, respectively) measured 2 and 4 weeks after acute elevation to 50 mm Hg. CONCLUSIONS. Acute IOP elevation in the rodent eye causes rapid, reversible posterior deformation of the ONH and thinning of the peripapillary retina, with only minimal retinal thinning beyond 5° of the ONH. No permanent changes in peripapillary retinal or RNFL thickness (for up to 1 month of follow-up) were caused by 60 minutes of IOP elevation to 50 mm Hg.

Purpose: To investigate the biomechanical response to IOP elevation of normal monkey eyes using eye-specific, three-dimensional (3-D) finite element (FE) models of the ONH that incorporate lamina cribrosa (LC) microarchitectural information., Methods: A serial sectioning and episcopic imaging technique was used to reconstruct the ONH and peripapillary sclera of four pairs of eyes fixed at 10 mm Hg. FE models were generated with local LC material properties representing the connective tissue volume fraction (CTVF) and predominant LC beam orientation and used to simulate an increase in IOP from 10 to 45 mm Hg. An LC material stiffness constant was varied to assess its influence on biomechanical response., Results: Strains and stresses within contralateral eyes were remarkably similar in both magnitude and distribution. Strain correlated inversely, and nonlinearly, with CTVF (median, r (2) = 0.73), with tensile strains largest in the temporal region. Stress correlated linearly with CTVF (median r(2) = 0.63), with the central and superior regions bearing the highest stresses. Net average LC displacement was either posterior or anterior, depending on whether the laminar material properties were compliant or stiff., Conclusions: The results show that contralateral eyes exhibit similar mechanical behavior and suggest that local mechanical stress and strain within the LC are correlate highly with local laminar CTVF. These simulations emphasize the importance of developing both high-resolution imaging of the LC microarchitecture and next-generation, deep-scanning OCT techniques to clarify the relationships between IOP-related LC displacement and CTVF-related stress and strain in the LC. Such imaging may predict sites of IOP-related damage in glaucoma.

Purpose: To characterize the trabeculated connective tissue microarchitecture of the lamina cribrosa (LC) in terms of total connective tissue volume (CTV), connective tissue volume fraction (CTVF), predominant beam orientation, and material anisotropy in monkeys with early experimental glaucoma (EG)., Methods: The optic nerve heads from three monkeys with unilateral EG and four bilaterally normal monkeys were three dimensionally reconstructed from tissues perfusion fixed at an intraocular pressure of 10 mm Hg. A three-dimensional segmentation algorithm was used to extract a binary, voxel-based representation of the porous LC connective tissue microstructure that was regionalized into 45 subvolumes, and the following quantities were calculated: total CTV within the LC, mean and regional CTVF, regional predominant beam orientation, and mean and regional material anisotropy., Results: Regional variation within the laminar microstructure was considerable within the normal eyes of all monkeys. The laminar connective tissue was generally most dense in the central and superior regions for the paired normal eyes, and laminar beams were radially oriented at the periphery for all eyes considered. CTV increased substantially in EG eyes compared with contralateral normal eyes (82%, 44%, 45% increases; P<0.05), but average CTVF changed little (-7%, 1%, and -2% in the EG eyes). There were more laminar beams through the thickness of the LC in the EG eyes than in the normal controls (46%, 18%, 17% increases)., Conclusions: The substantial increase in laminar CTV with little change in CTVF suggests that significant alterations in connective and nonconnective tissue components in the laminar region occur in the early stages of glaucomatous damage.

We report the case of a 39-year old male patient who presented with anaphylactoid shock and diffuse bleeding with prolonged activated partial thromboplastin time at the emergency room. The diagnosis of aggressive mastocytosis was suspected and then confirmed by raised tryptase level and mastocytic infiltration of the bone marrow. The outcome was favorable with supportive measures, antihistamine drugs, and imatinib mesylate.

Confocal microscopes (CM) are routinely used for building 3-D images of microscopic structures. Nonideal imaging conditions in a white-light CM introduce additive noise and blur. The optical section images need to be restored prior to quantitative analysis. We present an adaptive noise filtering technique using Karhunen-Loéve expansion (KLE) by the method of snapshots and a ringing metric to quantify the ringing artifacts introduced in the images restored at various iterations of iterative Lucy-Richardson deconvolution algorithm. The KLE provides a set of basis functions that comprise the optimal linear basis for an ensemble of empirical observations. We show that most of the noise in the scene can be removed by reconstructing the images using the KLE basis vector with the largest eigenvalue. The prefiltering scheme presented is faster and does not require prior knowledge about image noise. Optical sections processed using the KLE prefilter can be restored using a simple inverse restoration algorithm; thus, the methodology is suitable for real-time image restoration applications. The KLE image prefilter outperforms the temporal-average prefilter in restoring CM optical sections. The ringing metric developed uses simple binary morphological operations to quantify the ringing artifacts and confirms with the visual observation of ringing artifacts in the restored images.

Patients with mild/moderate hemophilia A (MHA) may develop inhibitors to factor VIII (FVIII). In this condition, FVIII clotting activity (FVIII:C) baseline levels may remain stable for some patients, but may be reduced to less than 0.01 U/mL for others. Several risk factors for the development of inhibitors in MHA have been proposed. Genetic factors, such as mutations in the FVIII gene, may play a central role; however, other influences, such as intensive treatment with FVIII products, may also be important. Optimal treatment regimens have yet to be determined, not only for the eradication of inhibitors, but also for the management or surgical prophylaxis of hemorrhages associated with this condition. Several treatment options for the control of bleeding in patients with MHA and inhibitors (MHAI) are currently available, and the choice of therapeutic strategy should be given careful consideration; some treatments may produce an anamnestic response, thus delaying the return to FVIII:C baseline levels and adversely affecting the duration of the severe bleeding phenotype. To increase our knowledge of MHAI, a retrospective collection of data is currently being performed among hemophilia centers in France and Belgium. Based on five examples of patients with MHAI collated from preliminary study data, we illustrate the impact on inhibitor outcome of the therapeutic choices used to treat bleeding episodes in these patients.

The purpose is to measure the costs of the new national breast cancer screening programme in France and to compare these with those of the previous programme in the Bouches-du-Rhône district. Direct screening costs and costs related to diagnosis and assessment were collected. Costs are presented by screening period, by organisms involved in the screening program and by corresponding phase within the screening process. The total cost of the screening program total cost has increased from 5587487 euros to 9345469 euros between the two campaigns. The main reasons are the investment costs in the new screening program, the increase in the target population and the increased fee for programs. This study presents a first estimate of the costs related to the new national breast cancer screening program. Results of this study may help to guide future decisions on the further development of breast cancer screening in France.

Coronary surgery with diabetes and severe hemophilia B is a challenging situation requiring specific and adequate therapeutic considerations. We herein report the case of a 52-year-old diabetic patient with severe factor IX deficiency and impaired ventricular function, who was scheduled for myocardial revascularization because of exertional angina and recurrent myocardial infarctions following infusions of factor IX concentrate. The patient underwent a successful off-pump direct myocardial revascularization with neither hemorrhagic nor thrombotic complications. Hematological protocols and operative strategies are assessed and discussed.

This retrospective, open-label, non-comparative study evaluated continuous infusion of recombinant factor VIII (ReFacto), B-domain deleted recombinant FVIII (BDDrFVIII), in patients with haemophilia A undergoing surgery and requiring >5 consecutive days of treatment. Sixteen patients from eight centres underwent a total of 20 procedures. Haemostatic outcome was assessed as 'excellent' or 'good' in 75% of procedures, and target FVIII:C levels were maintained throughout the continuous infusion period. The reported volume of blood loss during surgery was also within the normal range for non-haemophilic patients for the type of surgery performed. Red blood cell transfusions were required to balance excessive blood loss during BDDrFVIII continuous infusion in eight (40%) procedures (seven patients), five with bleeding or requiring volume replacement and three to treat anaemia secondary to blood loss. Non-serious adverse events considered by investigators as possibly or probably related to BDDrFVIII continuous infusion were infrequent (n = 5) considering the duration of treatment (n =239 cumulative days of continuous infusion), and all of these were mild-to-moderate in severity. No thromboembolic complications were reported except for one case of thrombophlebitis occurring at the infusion site. Only two patients (four events) experienced serious adverse bleeding; BDDrFVIII was otherwise well-tolerated. These data show that continuous infusion of BDDrFVIII provides reliable haemostasis and is an effective and well-tolerated regimen for patients with haemophilia A undergoing surgery.

Tissue factor pathway inhibitor (TFPI) is of major importance in regulating the coagulation triggering effects of tissue factor. An association between TFPI deficiency and thrombosis has still not been clearly demonstrated. We evaluated the anticoagulant activity of exogenous TFPI added either to the plasma of patients with venous thrombosis (n = 118) or to the plasma of healthy controls similar in terms of mean age and sex ratio (n = 107). A poor anticoagulant response to TFPI, defined as TFPI resistance, was observed in 4.7% of controls and in 11.0% of patients. TFPI resistance was associated with an almost threefold increase in the risk of thrombosis and could therefore represent a novel hemostatic risk factor for venous thrombosis.

The aim of the study was to assess the respective roles of the half-life of elimination of oral anticoagulants and patient education as causes of instability of anticoagulation level in patients on oral anticoagulant therapy. Patients were randomised to receive either warfarin (long half-life) or acenocoumarol (short half-life) and either intensive or standard education, according to a factorial design. Instability of oral anticoagulant therapy was evaluated by the percentage of INRs and the time within the target range, and the variability between successive measurements. Compliance was assessed by means of electronic pill bottles. Eighty-six patients were included. Apart from the variability index, instability was similar between groups. Correlations between compliance and instability were observed only in the acenocoumarol group. No difference was found between the education groups. In patients starting oral anticoagulant therapy, dose determination may be the most important factor contributing to instability.

Excessive apoptosis is implicated in the pathogenesis of myelodysplastic syndromes (MDS). We assessed by flow cytometry the expression of several members of the Bcl-2 family in bone marrow mononuclear cells (BMMNC) of 168 MDS samples at diagnosis. The proteins studied were Bcl-2, Bcl-xL (anti-apoptotic), Bax, Bad, Bak, and Bcl-xS (pro-apoptotic). The percentage of BMMNC expressing Bcl-2 and Bcl-xL was higher in refractory anemia with excess of blasts (RAEB), RAEB in transformation (RAEB-T), and chronic myelomonocytic leukemia (CMML) than in refractory anemia (RA) and RA with ringed sideroblasts (RAS). Conversely pro-apoptotic proteins Bad, Bak, and Bcl-xS were detected in a higher percentage of cells in RA and RAS. RA and RAS were associated with an increased Bcl-xS/Bcl-xL ratio. The expression of anti-apoptotic proteins was also correlated with that of CD34 and P170 and with the percentage of blast cells. Two-color analyses demonstrated that CD34 and Bcl-2 were usually expressed in the same cells. No significant correlation was found with cytogenetic abnormalities. Higher expression of pro-apoptotic Bcl-2-family proteins (Bak, Bad, Bcl-xS) and higher Bcl-xS/Bcl-xL ratio were associated with longer survival and decreased risk of leukemic transformation in univariate analysis, whereas expression of anti-apoptotic proteins was associated with decreased survival. Consequently Bcl-2 proteins expression was well correlated with the International Prognostic Scoring System (IPSS). Our data confirm that the control of apoptosis is deregulated in MDS cells. Moreover, the study of markers such as CD34 (or Bcl-2), Bcl-xL, and Bcl-xS provides additional prognostic information., (Copyright 2002 Wiley-Liss, Inc.)

Three new flavonol malonylrhamnosides, 3-O-(4"-O-malonyl)-alpha-L-rhamnopyranosides of mearnsetin, myricetin and quercetin respectively, together with the corresponding mearnsitrin, myricitrin, quercitrin and the 4-O-methyl phloracetophenone 2-O-beta-D-glucopyranoside, were isolated from the leaves of Ribes alpinum and fully characterized by spectrocopic methods including 2D NMR.

Factor VII (FVII) deficiency is a rare haemorrhagic condition, normally inherited as an autosomal recessive trait, in which clinical presentation is highly variable and correlates poorly with laboratory phenotype. The FVII (F7) gene was sequenced in 48 unrelated individuals with FVII deficiency, yielding a total of 23 novel lesions including 15 missense mutations, 2 micro-deletions, 5 splice junction mutations and a single base-pair substitution in the 5' untranslated region. Family studies were performed in order to distinguish the contributions of individual mutant F7 alleles to the clinical and laboratory phenotypes. Specific missense mutations were evaluated by molecular modelling in the context of the FVIIa-tissue factor crystal structure. Single base-pair substitutions in splice sites and the 5' untranslated region were studied by in vitro splicing assay and luciferase reporter gene assay, respectively. All probands were also typed for four previously reported F7 polymorphisms. In the majority of cases of FVII deficiency studied here, consideration of both mutational and polymorphism data permitted the derivation of plausible explanations for the FVII activity and antigen levels measured in the laboratory. Inter-familial variation in FVII activity and the antigen levels of heterozygous relatives of probands was found to be significantly higher than intra-familial variation, consistent with the view that the nature of the F7 gene lesion(s) segregating in a given family is a prime determinant of laboratory phenotype. Although no relationship could be discerned between laboratory phenotype and polymorphism genotype, the frequencies of the A2 and M2 polymorphic alleles were significantly higher in the FVII-deficient individuals tested than in controls. This suggests that the presence of these alleles may have served to increase the likelihood of pathological F7 gene lesions coming to clinical attention.

Objective: To evaluate the effectiveness and the safety of danaparoid sodium in the treatment of critically ill patients with standard unfractionated heparin-induced thrombocytopenia (HIT) or low-molecular-weight HIT., Setting: University hospital., Patients and Methods: Retrospective analysis of 42 consecutive critically ill patients who were admitted for HIT between October 1992 and February 1997 and were treated either with therapeutic or prophylactic doses of danaparoid sodium., Results: Among the 26 patients treated with therapeutic doses, neither new thrombotic complications nor thrombosis extension was clinically suspected. Two deaths were directly related to lower limb acute arterial thrombosis associated with HIT. Two major hemorrhagic complications were observed when aspirin in addition to danaparoid sodium was administered. When danaparoid sodium was used in prophylactic doses (20 courses of treatment) to prevent either postsurgical or medical thrombotic complications, no thrombotic event was observed. No death related to HIT or danaparoid sodium treatment was observed. One aggravation of a postsurgical cerebral lesion was observed. During danaparoid sodium treatment, a persistence or a recurrence of thrombocytopenia was observed in 6.5% of patients without thrombotic complications., Conclusion: Danaparoid sodium appears to be an efficient and safe treatment in critically ill patients with HIT. The concomitant use of aspirin in addition to danaparoid sodium seems to represent an important additional hemorrhagic risk that should be avoided in patient management.

Objective: To investigate the role of activated protein C resistance (APCR, factor V Leiden) in coronary artery thrombosis., Methods: The prevalence of APCR and of congenital deficiencies of antithrombin III, protein C, protein S, plasminogen, and factor XII was investigated in adult patients under 45 years of age with acute myocardial infarction. The results were compared with those of a group of 53 age and sex matched control subjects., Results: Among 75 patients under the age of 45 years who were admitted from November 1994 to April 1996 for acute myocardial infarction, 22 (29.3%) had normal coronary arteriography (group I) and 53 (70.7%) had significant coronary artery disease (group II). Inherited thrombophilia was more often found in group I (4/22, 18.2%) than in group II (4/53, 7.5%) but the difference was not significant (F test: p = 0.22). The prevalence of APCR was 9.1% (2/22) in group I, 3.8% (2/53) in group 2 (p = 0.57), and 3.8% (2/53) in the normal control group (p = 0.57)., Conclusions: The prevalence of congenital thrombophilias, including APCR, does not seem to be increased in young patients with myocardial infarction and normal coronary angiograms, compared with young patients with coronary atherosclerosis and with normal control subjects. However, the statistical power of the study is too low to detect a significant difference and these results are published to allow a meta-analysis of this problem in the future.

Objectives: Heparin-induced thrombocytopenia is a relatively common and potentially serious adverse complication of heparin treatment. After heparin withdrawal initiation of an alternative anticoagulant is often indicated. Org 10172 or Orgaran is a mixture of several non-heparin low molecular weight glycosaminoglycans with proven antithrombotic efficacy. Unlike low molecular weight heparins, Org 10172 has a low cross reaction rate (about 10%) with the heparin-dependent antibody., Methods: We present nine patients with heparin induced thrombocytopenia. Org 10172 was prescribed to treat or to prevent a thromboembolic event., Results: Seven patients required further parenteral anticoagulant at diagnosis of heparin-induced thrombocytopenia. Org 10172 was given at prophylactic doses for three patients with a high thrombosis risk and at therapeutic doses for four patients who presented either a venous or an arterial thrombosis related to thrombocytopenia. Two patients presented heparin-induced thrombocytopenia four to six years earlier and needed a parenteral anticoagulation treatment in a post-operative period., Conclusion: For the nine patients, Org 10172 was a safe and effective antithrombotic treatment. However, strict monotoring of the platelet count is absolutely mandatory during Org 10172 therapy.

In an effort to further understand Glanzmann thrombasthenia (GT) 3 patients from 2 different families were studied. After biochemical and immunological analysis these patients were classified as type I. We observed in the first family a new restriction site for Stu I in exon II of the glycoprotein (GP) IIIa gene caused by a homozygous nonsense mutation: 62 Arg to stop codon. The parents were heterozygotes for this mutation. We found in the second family a previously described nonsense mutation: 584 Arg to stop codon in exon 17 of the GPIIb gene. The father and his two affected sons were heterozygous for this genetic defect. This mutation 62 Arg to stop codon is a new description of a genetic defect associated with GT. Furthermore, the discovery of the same mutation in 3 affected families from different ethnic groups raises the possibility of either a hot spot mutation in the CG dinucleotide region of GPIIb gene, or an ancient mutant allele present in diffuse populations at a relatively high frequency.

Low molecular weight heparin (LMWH) is currently prescribed for the treatment of deep vein thrombosis at the dose of 100 IU antiXa/kg twice daily or at a dose of 175 IU antiXa/kg once daily with a similar efficacy. We decided to study the chrono-pharmacology of curative dose of LMWH once daily administrated according to the one previously described with unfractionated heparin (UFH). Ten healthy volunteers participated in an open three-period crossover study according to three 24 h cycles, separated by a wash-out interval lasting 7 days: one control cycle without injection, two cycles with subcutaneous injection of 200 IU antiXa/kg of Dalteparin (Fragmin) at 8 a.m. or at 8 p.m. Parameters of heparin activity were analysed as maximal values and area under the curve. Activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT) and tissue factor pathway inhibitor (TFPI) were higher after 8 p.m. injection than after 8 a.m. injection (p < 0.05) while no chrono-pharmacological variation of anti factor Xa (AXa) activity was observed. Thus the biological anticoagulant effect of 200 IU antiXa/kg of Dalteparin seems to be higher after an evening injection than after a morning injection. A chrono-therapeutic approach with LMWH, as prescribed once daily, deserves further investigation since our results suggest that a preferential injection time may optimise the clinical efficacy of these LMWH.

Missense mutations, three of them novel (Gly47-->Cys, Arg178-->Pro, Ala259-->Thr), were found in the protein C genes of four patients with inherited protein C deficiency associated with venous thrombosis. Comparison with the phenotypic effects of mutations in the analogous residues of factor IX and the use of a molecular model of protein C provided explanations as to how these lesions might alter either the structure, function or stability of the protein C molecules encoded.

The local and systemic fibrinolytic response to the placing of elastic compression in healthy volunteers was studied to determine whether this method of preventing venous thromboembolism has any profibrinolytic effect, as previously demonstrated with intermittent pneumatic compression. Variations in the major parameters of fibrinolysis (euglobulin lysis time, t-PA antigen, PAI-1 antigen, PAI-1 activity) were studied in an open randomized cross-over study in 21 healthy volunteers, in which three types of treatment were tested for periods of 24 h each (without elastic compression, elastic compression of an upper limb, elastic compression of the lower limbs). Four blood samples were taken from the upper limb during each period (at 08:00 h, 10:00 h, 18:00 h, 08:00 h on the following day). The placing of elastic compression did not cause any statistically significant change in the four parameters tested between the three types of treatment. In contrast, circadian rhythm was confirmed for all the fibrinolytic factors studied with a minimal fibrinolytic activity in the morning and a maximal activity in the evening. Elastic compression does not seem to have any profibrinolytic effect in healthy volunteers but other studies are needed in patients before a definitive conclusion can be reached.

We report six cases of protein S deficiency secondary to varicella. Five cases were complicated by thrombotic and vascular events, namely purpura fulminans and necrotic vasculitis, deep vein thrombosis and stroke. Two cases were associated with protein C deficiency and one case revealed a heterozygous factor XII deficiency. The underlying mechanism of this acquired protein S deficiency is unclear but could be related to a direct effect of zoster virus.

Pentosan polysulfate is a low-molecular-weight sulfated polysaccharide used as an antithrombotic drug. We present two patients who developed thrombocytopenia and venous thrombosis during treatment with pentosan polysulfate. The relationship between pentosan polysulfate and thrombocytopenia is supported by platelet aggregation and serotonin release tests. In the light of the literature and our two cases, it appears that pentosan polysulfate alone as standard heparin and low-molecular-weight heparin can induce thrombocytopenia and thrombosis. Platelet counts should therefore be periodically monitored during pentosan polysulfate treatment. In the case of pentosan polysulfate-induced thrombocytopenia, it seems that heparin or low-molecular-weight heparin should not be instituted during the acute phase even if platelet aggregation studies are negative, because of their low sensitivity. After remission of thrombocytopenia, whether or not glycosaminoglycans can be reinstituted, at least temporarily, after antibody had disappeared is still an open question.

Trisomy 12 is the most common cytogenetic abnormality in chronic lymphocytic leukaemia (CLL) and may be a prognostic indicator. In the present study, fluorescence in situ hybridization (FISH) is shown to be a method of choice for detection of trisomy 12 in interphase cells. Seventy-five cases of B-cell CLL were analysed with a chromosome 12 specific alpha satellite DNA probe and results compared with those from cytogenetic analysis. FISH showed the three hybridization spots characteristic of trisomy 12 in 32/75 patients (42.6%). Sixty-three patients were also studied by conventional cytogenetics: failure in 7 cases, normal karyotype in 28, trisomy 12 in 9 (14.3%) and in 19 cases abnormalities other than trisomy 12. In these same 63 patients, trisomy 12 was detected on 29 occasions by FISH (46%): in one case of failure by cytogenetic analysis, in 9 cases thought to have a normal karyotype, in 10 cases carrying abnormalities other than trisomy 12 and in all 9 cases showing trisomy 12 by conventional cytogenetic investigation. Correlation between trisomy 12 and the three stages of the Binet classification indicated an increasing proportion of trisomy 12 from stage A to stage C. It is concluded that fluorescence in situ hybridization is a powerful and sensitive technique for detection of trisomy 12 in CLL and although more cases will be required to confirm a correlation between the incidence of trisomy 12 and the stage of the disease, this link could be important from a prognostic point of view.

This study evaluated the effect of injection time on pharmacodynamic of a single subcutaneous bolus of nadroparine (7500 anti-Xa IC U) evaluated by anti-Xa activity (Hepaclot and Heptest) and by activated partial thromboplastin time (APTT by auto PTT reagent). 10 healthy male volunteers were studied at 4 different 24 hours periods with 4 different injection times (6 am, 12 am, 6 pm, 12 pm) and with a one week wash-out period between each period. No chronopharmacological variation of the anti-Xa activity evaluated by Hepaclot was found. However the anti-Xa activity evaluated by Heptest was higher at the sixth hour after 12 am injection (p = 0.0022). No difference on APTT values was observed whatever the injection time. So the injection time of nadroparine has a weak influence on anti-Xa activity and no effect on APTT; Before to conclude on the lack of chronopharmacological effect of nadroparine, it seems necessary to evaluate such a possibility with higher dosage, with sick and older subjects.

Variant translocations (2;18 and 18;22) are described in this review. The chromosomal and molecular findings of these translocation of BCL2 and their effect on possible BCL2 gene activation is discussed. Unanswered questions still remain and these include why this is so rare compared to the 25% incidence recorded for translocations in Burkitt's lymphoma. Further studies are obviously still needed in order to determine the true frequency of these findings and their distribution in the various B-cell disorders.

A 3-year old child was admitted for a third relapse of nephrotic syndrome associated with intracranial hypertension related to dural sinus thrombosis (tomodensitometry). The treatment consisted in the association of low dose heparin and fresh frozen plasma. After a 3 year-follow-up, there was no neurologic sequelae, and the nephrotic syndrome was on complete remission. The radiologic features and the management of sinus thrombosis are discussed.

We report a 56-year-old male patient with refractory anemia with excess of blasts in transformation (RAEB-T) who had an ins(8;3)(q24;q21q26) as the sole chromosome abnormality in bone marrow (BM) cells. The findings of disturbed thrombocytopoiesis with numerous micromegakaryocytes suggest that it could be a variant of the classic ins(3;3)(q26;q21q26) described in hematologic malignancies with abnormal thrombopoiesis.

Alexander disease, or hypoproconvertinemia is a rare autosomic recessive coagulation disorder. The features include familial and/or personal history of bleeding, with an abnormal prothrombin period and a normal activated partial thromboplastin period. Coagulation and genetic studies allow subclassification with prognosis incidence for this disease. The authors report on a case of one family with Alexander disease.

In order to compare the effects of interferon versus hydroxyurea for the treatment of chronic myelogenous leukaemia (CML), 58 CML patients, having received no previous treatment, were randomized into two treatment groups (hydroxyurea or interferon) for an open multicentre study from 1 May 1987 until 1 July 1990. Fifty patients were evaluable: 24 in the interferon group and 26 in the hydroxyurea group. Haematological response was obtained in 16/24 interferon-treated patients and 23/26 hydroxyurea patients. Failure to obtain haematological remissions occurred in eight of 24 interferon-treated patients and in three of 26 hydroxyurea patients. Four interferon-treated patient failures and one hydroxyurea-treated failure were due to drug intolerance. Progression occurred in one interferon-treated patient and in three patients given hydroxyurea. Fourteen of 16 patients in the interferon group and 17/23 in the hydroxyurea group continue on study and show no progression.

Like other workers, we found gender differences in auditory-evoked brainstem response latencies. For the time being, this intersubject variability remains unexplained. This difference was observed only for wave V latency, and delta I-V is longer in men than in women, whereas delta I-III is identical. The absence of functional differences in the lower portion of the auditory tract is confirmed by both auditory-evoked brainstem potentials and stapedius reflex latency. Wave V latency and the influence of technical and anatomical factors are discussed as well.