Background

Cold agglutinin disease is a rare disorder characterized by an autoimmune hemolytic anemia occurring at low temperatures. Physical examination findings, often limited to acrocyanosis, are combined with a thermal amplitude test to help establish the diagnosis. Thermal amplitude testing determines the highest temperature at which the cold agglutination will occur and is an important parameter in diagnosing cold agglutinin disease.

Case presentation

Here we describe a 57-year-old white man of German and Nicaraguan descent with known chronic cold agglutinin disease who presented to our ophthalmology clinic for evaluation of a cataract. During routine cataract surgery, the lowered temperature of the conjunctiva from intermittent flow of balanced salt solution at room temperature induced a cold agglutination reaction in conjunctival vessels easily visible under a surgical microscope.

Conclusions

To the best of our knowledge, this method of demonstrating cold agglutinin disease has not been described in the literature and could easily be performed utilizing an ordinary slit lamp. This method could be used as an alternative and rapid screening method for cold agglutinin disease.

Purpose

A critical biological function of retina pigment epithelium (RPE) cells is phagocytosis of photoreceptor outer segment (POS) disc membranes. Mitochondrial damage and dysfunction are associated with RPE cells of age-related macular degeneration (AMD) retinas. In this study, we use a transmitochondrial cybrid model to compare the phagocytic properties of RPE cells that contain AMD mitochondria versus age-matched normal mitochondria and their response to treatment with anti-vascular endothelial growth factor (VEGF) drugs: bevacizumab, ranibizumab, and aflibercept.

Methods

Cybrids, which are cell lines with identical nuclei but mitochondria (mt) from different subjects, are created by fusing mtDNA depleted ARPE-19 cells with platelets from AMD or age-matched normal patients. AMD (n = 5) and normal (n = 5) cybrids were treated with 1 μm fluorescent latex beads (1.52 × 10⁷ beads/mL) and either 2.09 μM of bevacizumab, 2.59 μM of ranibizumab, or 5.16 μM of aflibercept. These doses of anti-VEGF drugs are equivalent to intravitreal injections given to AMD patients with choroidal neovascularization. Flow cytometry was performed using the ImageStreamX Mark II to assess phagocytic bead-uptake. The average fold values for bead-uptake and SEM were calculated using GraphPad Prism software.

Results

Normal cybrids showed decreased bead-uptake with a fold value of 0.65 ± 0.103 (p = 0.01) after treatment with bevacizumab, 0.80 ± 0.034 (p = 0.0003) with ranibizumab, and 0.81 ± 0.053 (p = 0.007) with aflibercept compared to the untreated normal cybrids (baseline fold of 1). The bevacizumab-treated, ranibizumab-treated, and aflibercept-treated AMD cybrids had decreased bead-uptake with a fold value of 0.71 ± 0.061 (p = 0.001), 0.70 ± 0.101 (p = 0.02), and 0.74 ± 0.125 (p = 0.07), respectively, compared to the untreated AMD cybrids (baseline fold of 1).

Conclusions

Our initial findings showed that when treated with bevacizumab and ranibizumab, both AMD cybrids and age-matched normal cybrids had a significant decrease in bead-uptake. A similar decrease in bead-uptake was found in normal cybrids treated with aflibercept and while the AMD values trended lower, they were not significant. This data suggests that anti-VEGF drugs can cause loss of phagocytic function.