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. 2021 Mar 4;16(3):e0247846.
doi: 10.1371/journal.pone.0247846. eCollection 2021.

Non-invasive intradermal imaging of cystine crystals in cystinosis

Marya Bengali  1 , Spencer Goodman  1 , Xiaoying Sun  2 , Magdalene A Dohil  3 , Ranjan Dohil  4 , Robert Newbury  5 , Tatiana Lobry  1 , Laura Hernandez  1 , Corinne Antignac  6   7 , Sonia Jain  2 , Stephanie Cherqui  1
Affiliations

Non-invasive intradermal imaging of cystine crystals in cystinosis

Marya Bengali et al. PLoS One. .

Abstract

Importance: Development of noninvasive methodology to reproducibly measure tissue cystine crystal load to assess disease status and guide clinical care in cystinosis, an inherited lysosomal storage disorder characterized by widespread cystine crystal accumulation.

Objective: To develop an unbiased and semi-automated imaging methodology to quantify dermal cystine crystal accumulation in patients to correlate with disease status.

Design, setting and participants: 101 participants, 70 patients and 31 healthy controls, were enrolled at the University of California, San Diego, Cystinosis Clinics, Rady Children's Hospital, San Diego and at the annual Cystinosis Research Foundation family conference for an ongoing prospective longitudinal cohort study of cystinosis patients with potential yearly follow-up.

Exposures: Intradermal reflectance confocal microscopy (RCM) imaging, blood collection via standard venipuncture, medical record collection, and occasional skin punch biopsies.

Main outcomes and measures: The primary outcome was to establish an automated measure of normalized confocal crystal volume (nCCV) for each subject. Secondary analysis examined the association of nCCV with various clinical indicators to assess nCCV's possible predictive potential.

Results: Over 2 years, 57 patients diagnosed with cystinosis (median [range] age: 15.1 yrs [0.8, 54]; 41.4% female) were intradermally assessed by RCM to produce 84 image stacks. 27 healthy individuals (38.7 yrs [10, 85]; 53.1% female) were also imaged providing 37 control image stacks. Automated 2D crystal area quantification revealed that patients had significantly elevated crystal accumulation within the superficial dermis. 3D volumetric analysis of this region was significantly higher in patients compared to healthy controls (mean [SD]: 1934.0 μm3 [1169.1] for patients vs. 363.1 μm3 [194.3] for controls, P<0.001). Medical outcome data was collected from 43 patients with infantile cystinosis (media [range] age: 11 yrs [0.8, 54]; 51% female). nCCV was positively associated with hypothyroidism (OR = 19.68, 95% CI: [1.60, 242.46], P = 0.02) and stage of chronic kidney disease (slope estimate = 0.53, 95%CI: [0.05, 1.00], P = 0.03).

Conclusions and relevance: This study used non-invasive RCM imaging to develop an intradermal cystine crystal quantification method. Results showed that cystinosis patients had increased nCCV compared to healthy controls. Level of patient nCCV correlated with several clinical outcomes suggesting nCCV may be used as a potential new biomarker for cystinosis to monitor long-term disease control and medication compliance.

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Conflict of interest statement

I have read the journal’s policy and the authors of this manuscript have the following competing interests: Stephanie Cherqui is inventor on a patent entitled “Methods of treating mitochondrial disorders” (#20378-201301) and co-inventor on a patent entitled “Methods of treating lysosomal disorders” (#20378-202488). She is a cofounder, shareholder and a member of both the Scientific Board and Board of Directors of Stelios Therapeutics Inc. Stephanie Cherqui also serves as a member of the Scientific Review Board and Board of Trustees of the Cystinosis Research Foundation. The terms of this arrangement have been reviewed and approved by the University of California San Diego in accordance with its conflict of interest policies. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no other patents, products in development or marketed products associated with this research to declare.

Figures

Fig 1
Fig 1. Skin biopsies and intradermal confocal imaging highlight crystals in cystinosis patients.
A-C, Skin punch biopsies taken from 1–2 cm behind the mastoid region of the right ear of two cystinosis patients (A-B) and a healthy control (C). Skin biopsy samples were sectioned at 1 μm and then stained with Toluidine Blue to detect cystine crystals and H&E for structural evaluation. Arrows indicate crystals. Crystal counting and morphological measurements were conducted by a clinical pathologist. Scale bar toluidine blue = 50 μm. Scale bar H&E = 100 μm. White arrowhead indicate perivascular chronic inflammatory cells in the upper dermis (A). L = lymphocyte, F = fibroblast nucleus, Sq. Epi. = squamous cell epithelium, BV = blood vessel. D, Intradermal imaging methodology and sample patient image. Using a handheld RCM device, a 78 slice Zstack with step size of 2.8 μm was taken behind the left ear beginning within the epidermis. A representative single slice with arrows indicating crystals is shown, while complete patient and control Zstacks are provided in S1 and S2 Videos. Scale bar = 100 μm.
Fig 2
Fig 2. Automated 2D and 3D image analysis detects an increased crystallization in cystinotic skin.
A total of 83 image stacks were acquired from 70 cystinosis patients and 38 from 27 healthy controls. A, Workflow from initial selection of total crystals and skin structure to final 3D reconstruction of the papillary dermis region. Arrows indicate false-positive crystals which are eliminated due to overlap with skin structure. Full description of methodology may be found in S1 Fig and Supplementary Methods in (S1 File). B, Representative slices from raw and 2D analyzed intradermal confocal micrographs from a healthy control and patients. XY scatterplot displays the sum of crystal area normalized to total imaged region on the Y-axis vs. tissue depth on X-axis. Arrows indicate which slice is the sample image. C, XY scatterplot depicting mean crystal area +/- SD vs. tissue depth for grouped cystinosis patients vs. healthy controls. For subjects with multiple images, only the most recent encounter was included. Starred region indicates slices where patients have significantly higher crystal area. D, 3D reconstructions of healthy and patient crystal density exclusively in the papillary dermal region due to signal background in epidermis and hypodermis; see Results section “Crystal quantification by automated image analysis in 2D” for details. Representative videos are provided in S3 and S4 Videos. E, Boxplot comparing sum crystal volume in papillary dermis (nCCV) +/- SD between patients and controls. **** = P<0.001. All scale bars = 100 μm.
Fig 3
Fig 3. Focused case study investigating predictive potential of nCCV for two cystinosis patients.
A-B, Intradermal imaging and analysis in 2D and 3D for two cystinosis subjects: Subject 1, a 20 yr-old compliant male without a kidney transplant and compound heterozygous for the 57kb deletion and a frameshift mutation (A), and Subject 2, a 31 yr-old non-compliant patient, bearing the 57kb deletion in the homozygous state, who has received his first kidney transplantation at 14 years and second at 26 years of age (B). Scale bar = 100 μm. C, nCCV quantitation of Patients 1 and 2 for multiple years of acquisition. (D) Selected medical outcomes displaying subject 1 (blue star) and subject 2 (red star) compared to the full set of patients (each dot represents a patient) for various symptoms during the most recent year of acquisition, n = 36 for TSH, n = 31 for PTH, n = 41 for BP Systolic, and n = 37 for BMI.
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