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Fast and easy visualization method of impression cytology probe with microbiota detection on the ocular surface

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Abstract

Introduction Impression cytology is non-or minimally invasive technique used for ocular surface examination. This technique is in constant expansion due to its advantages and high diagnostic potential. Usually the conjunctival cavity and its microbiota are examined by light microscopy or bacteriological culture methods. However, limitations of the light microscopy method do not allow to visualize properly the bacterial component of the probe. After that, microbiological culture methods are time consuming and some types of bacteria can't be cultivated in standard conditions, what can lead to false negative results. Identification of the pathogen is crucial on behalf of choosing an antibacterial therapy. This challenge can be solved by scanning electron microscopy. At the same time, conventional sample preparation is quite a long process. We aimed to use a new fast two-step lanthanoid contrasting method with subsequent visualization on scanning electron microscope. Materials & Methods 12 patients participated in the study. 6 of them had specific signs of infectious inflammatory process on the ocular surface. Other patients didn't suffer from ocular surface diseases. Topical anesthesia was not required, because flexible plastic cover slips didn't cause any discomfort to the patients. The specimen preparation included 2 stages: treatment with neodymium and with plumbum (BioREE-B staining kit). Further, these specimens were examined on the scanning electron microscope. Results In visualized samples we found squamous epithelial cells, mucosal components and microbiota. Epithelial cells seemed to enter the apoptotic process (karyopicnosis and cytoplasm vacuolation). In two out of six cases with supposed infectious process increased density of coccomorphic organisms compared with "healthy" donors was detected. Sometimes it was possible to visualize intercellular contacts. Conclusion It is the first time, when pictures of ocular surface were obtained so fast and with so high resolution. New contrasting method with neodymium and plumbum treatment enable to give the morphological assessment of the most superficial layers. Furthermore, quality of the images allows to evaluate not only functional status of corneal cells, but also microbiota activity.
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Fast and easy visualization method of impression cytology probe
with microbiota detection on the ocular surface
Yugay, N.Y. (Nikolay)1, Novikov, N.I. (Ivan)2, Subbot, S.A. (Anastasia) PhD2, Khalatyan, K.S. (Anait)3
1 I.M. Sechenov First Moscow State Medical University (Sechenovskiy University), Ophthalmology, Moscow,
Russian Federation
2 Research Institute of Eye Diseases, Laboratory of Basic Research in Ophthalmology, Moscow, Russian
Federation
3 Research Institute of Eye Diseases, Ophthalmology, Moscow, Russian Federation
Introduction
Impression cytology is non- or minimally invasive technique used for ocular surface examination.
This technique is in constant expansion due to its advantages and high diagnostic potential.
Usually the conjunctival cavity and its microbiota are examined by light microscopy or
bacteriological culture methods. However, limitations of the light microscopy method do not
allow to visualize properly the bacterial component of the probe. After that, microbiological
culture methods are time consuming and some types of bacteria can’t be cultivated in standard
conditions, what can lead to false negative results. Identication of the pathogen is crucial on
behalf of choosing an antibacterial therapy. This challenge can be solved by scanning electron
microscopy. At the same time, conventional sample preparation is quite a long process. We
aimed to use a new fast two-step lanthanoid contrasting method with subsequent visualization
on scanning electron microscope.
Materials & Methods
12 patients participated in the study. 6 of them had specic signs of infectious inammatory
process on the ocular surface. Other patients didn’t suer from ocular surface diseases. Topical
anesthesia was not required, because exible plastic cover slips didn’t cause any discomfort to
the patients. The specimen preparation included 2 stages: treatment with neodymium and with
plumbum (BioREE-B staining kit). Further, these specimens were examined on the scanning
electron microscope.
Results
In visualized samples we found squamous epithelial cells, mucosal components and microbiota.
Epithelial cells seemed to enter the apoptotic process (karyopicnosis and cytoplasm vacuolation).
In two out of six cases with supposed infectious process increased density of coccomorphic
organisms compared with “healthy” donors was detected. Sometimes it was possible to visualize
intercellular contacts.
Conclusion
It is the rst time, when pictures of ocular surface were obtained so fast and with so high
resolution. New contrasting method with neodymium and plumbum treatment enable to give
the morphological assessment of the most supercial layers. Furthermore, quality of the images
allows to evaluate not only functional status of corneal cells, but also microbiota activity.
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