Let's Get Rolling: Precise Control of Microfluidic Assay Conditions to Recapitulate Selectin-Mediated Rolling Interactions of the Leukocyte Adhesion Cascade.

The leukocyte adhesion cascade governs the recruitment of circulating immune cells from the vasculature to distal sites. The initial adhesive interactions between cell surface ligands displaying sialyl-LewisX (sLeX) and endothelial E- and P-selectins serve to slow the cells down enough to interact more closely with the surface, polarize, and exit into the tissues. Therefore, precise microfluidic assays are critical in modeling how well immune cells can interact and "roll" on selectins to slow down enough to complete further steps of the cascade. Here, we present a systematic protocol for selectin mediated rolling on recombinant surfaces and endothelial cell monolayers on polyacrylamide gels of varying stiffness. We also describe step-by-step the protocol for setting up and performing the experiment and how to analyze and present the data collected. This protocol serves to simplify and detail the procedure needed to investigate the initial selectin-mediated interactions of immune cells with the vasculature. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparing dishes for cell rolling experiments Basic Protocol 2: Fabrication of polyacrylamide gels for cell rolling experiments Alternate Protocol 1: Protein conjugation with N6 linker Alternate Protocol 2: HUVEC culturing for monolayers Basic Protocol 3: Conducting cell rolling experiments on polyacrylamide gels Basic Protocol 4: ImageJ analysis of cell rolling movies Basic Protocol 5: Quantification of Fc site density on a surface (e.g., for Fc chimeras).

Comparison of Pre- and Post-DMEK Keratometry and Total Keratometry Values for IOL Power Calculations in Eyes Undergoing Triple DMEK.

PURPOSE: To evaluate prediction accuracy of pre- and post-DMEK keratometry (K) and total keratometry (TK) values for IOL power calculations in Fuchs endothelial corneal dystrophy (FECD) eyes undergoing DMEK with cataract surgery (triple DMEK). METHODS: Retrospective cross-sectional multicenter study of 55 FECD eyes (44 patients) that underwent triple DMEK between 2019 and 2022 between two centers in USA and Europe. Swept-source optical coherence tomography biometry (IOLMaster 700) was used for pre- and post-DMEK measurements. K and TK values were used for power calculations with ten formulae (Barrett Universal II (BUII), Castrop, Cooke K6, EVO 2.0, Haigis, Hoffer Q, Hoffer QST, Holladay I, Kane, and SRK/T). Mean error, mean absolute error (MAE), standard deviation, and percentage of eyes within ±0.50/±1.00 diopters (D) were calculated. Studied formulae were additionally adjusted using a method published previously (IOLup1D Method), which increases the IOL power by 1D. While both eyes from the same patient were considered for descriptive statistics, we restricted to one eye per individual (44 eyes for statistical comparisons. RESULTS: MAEs for all formulae were lower for post-DMEK K and TK than pre-DMEK K and TK by an average of 0.24 and 0.47 D, respectively. The lowest MAE was 0.49 D for Kane using post-DMEK TK, and the highest MAE was 1.05 D for BUII using pre-DMEK TK. Most IOLup1D formulae had lower MAEs than pre-DMEK K and TK formulae. CONCLUSIONS: The IOLup1D Method should be used instead of pre-DMEK K and TK values for triple DMEK in FECD eyes. Using post-DMEK TK values for cataract surgery after DMEK provides better refractive accuracy than any of the three studied methods used for triple DMEK procedures.

Standard vs total keratometry for intraocular lens power calculation in cataract surgery combined with DMEK.

PURPOSE: To compare the prediction accuracy of standard keratometry (K) and total keratometry (TK) for intraocular lens (IOL) power calculation in eyes undergoing combined cataract surgery and Descemet membrane endothelial keratoplasty (triple DMEK). SETTING: Tertiary care academic referral center. DESIGN: Retrospective case series. METHODS: Review of 83 eyes (63 patients) that underwent triple DMEK between 2019 and 2021. Biometry measurements were obtained using a swept-source optical biometer (IOLMaster 700). 63 eyes were used for statistical analysis. Mean error, mean absolute error (MAE), SD, median absolute error, maximum absolute error, root mean squared prediction error, and the percentage of eyes within prediction errors of ±0.50 diopters (D) and ±1.00 D were calculated for 9 multivariate and third-generation formulas using K and TK values (Barrett Universal II, Yeo EVO 2.0, Cooke K6, Kane, Pearl-DGS, Haigis, Holladay 1, Hoffer Q, and SRK/T). Formulas were additionally tested by using the prediction for an IOL power 1 D below the IOL used (IOLup1D). RESULTS: For all formulas, MAE was lower for K than for TK by an average of 0.21 D. The lowest MAE value observed was 0.67 D for "adjusted" SRK/T using K, and the highest MAE values observed were 1.24 D and 1.24 D for nonadjusted Hoffer Q and Haigis using TK, respectively. Overall, lower MAE values were observed for multivariate formulas and SRK/T. CONCLUSIONS: In triple DMEK eyes, the prediction accuracy of K was higher than that of TK. The most accurate formulas were SRK/T and multivariate formulas using K with the IOLup1D adjustment.

Choroidal thickness in non-ocular Behçet's disease - A spectral-domain OCT study.

PURPOSE: To evaluate choroidal thickness in patients with non-ocular Behçet's disease (BD) using spectral domain optical coherence tomography (SD-OCT) and to compare the results to normal eyes. METHODS: In this retrospective observational comparative study, we collected OCT and clinical data from the charts of 4 patients (7 eyes) with BD who had been referred for a screening eye exam and had a normal ocular examination. Data from 9 healthy volunteers (17 eyes) were collected as age-matched controls. The choroid was manually segmented from volume OCT scans using custom Doheny Image Reading Center OCT grading software (3D-OCTOR). Main outcome measures were choroidal thickness and intensity were compared between eyes of patients with BD and those of healthy controls. RESULTS: Eyes of patients with non-ocular BD had significantly thinner mean central subfield choroidal thickness (227.5 ± 56.93 versus 306.85 ± 17.85, P = 0.04) and central subfield choroidal volume (0.18 ± 0.04 vs 0.24 ± 0.02, P = 0.005). There was no significant difference in mean choroidal thickness in the whole ETDRS grid or in mean choroidal intensity in the central subfield and the whole ETDRS grid between eyes of patients with non-ocular BD and those of controls. CONCLUSION: This study demonstrates that BD may have subclinical manifestations in the choroid, resulting in thinning of the choroid relative to normal eyes, even without overt signs of ocular involvement.