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Dr. Johan A. Hutauruk
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Specular Microscopy
Corneal anatomy
at a glance
Descemet’s membrane
Thick basal lamina secreted by endothelium
– Anterior banded 3 μm at birth and stable
– Posterior non-banded 2 μm at birth, increased to 10 μm in adults
• Mainly Type IV collagen, Type VIII and Laminin
• Dua’s Layer
• Just anterior to Descemet's membare
• A very thin and strong layer, 15 microns thick
• Function ?
Endothelium
• Single cell, 4-6 mm thick, total  400,000 cells
– At birth :3,500 to 6,000 cell /mm2
– Adult :1,400 to 2,500 cell /mm2
• Hexagonal, 20mm wide : amitotic
• Has a single primary cilium : function?
• Large nucleus
• Smolin G, Thoft RA. The Cornea, 3rd Ed. Boston : Little, Brown & Co., 1994
Endothelium
• Function :
– Effective barrier from the aqueous
– Maintain metabolic pump
– Maintain corneal transparency
Endothelium
Kaufman HE, Barron BA, McDonald MB. The Cornea. New York: Churchil Livingstone 1989
Endothelium
• Cytoplasm :
– Numerous mitochondria
– Smooth & rough ER
– Golgi apparatus
• Critical density : Less than 500 cells/mm2
– Not enough room on the lateral cell membrane for the metabolic
pump site  corneal edema
Endothelium
• Endothelial barrier :
– Permeable
– Tight junction very sensitive to calcium, cause the
junction to break down
– Also regulated by the cytoskeleton (F-action) of
endothelial cells
Endothelium
• Endothelial pump :
– Each cell pumps its own value every 5 minutes
– Na+ K+ ATPase
• Located in lateral cell membrane
• Glucose fueled
• 3 million pump sites per endothelial cells
– Not fluid pump
• Transport ions : Na, bicarbonate from stroma to aqueous
• Water follows passively
Smolin G, Thoft RA. The Cornea, 3rd Ed.
Boston : Little, Brown & Co., 1994
Endothelium
• Fuchs’ dystrophy :
• Endothelial secrete abnormal collagen
• Endothelial cells stretch
• Permeability increases
– Early stages : normal corneal thickness due to increase pump
rate
– Late stages : corneal edema
•Exhausted Na K pump
•Lateral cell membrane lack of room for additional pump site
VISITING PROFESSORSHIP PROGRAM : INASCRS JEC
OPTICAL PRINCIPLE OF SPECULAR MICROSCOPY
▸ Light striking a surface :
▸ Reflected
▸ Transmitted
▸ Absorbed
▸ Specular microscopy :
▸ Light is reflected specularly (mirror-like)
▸ Angle of reflection = angle of incidence
INTRODUCTION
SPECULAR MICROSCOPE
▸ First described by Maurice (1968)
▸ Modified by Laing, Bourne and
Kaufman: replacing the original water
immersion lens
ORIGINAL ARTICLE
CORNEAL
ENDOTHELIAL
CELL
MORPHOLOGY
• Cell Area ± S.D. (µm2)
• Cell Density (cells / mm2)
• Polymegethism (CV)
• Pleomorphism
(% hexagonal 6 sided)
Number: the number of analyzed cells
CD : cell density
AVG : Average cell area
SD: standard deviation of cell area
CV : Coefficient of variation of cell area
Max: maximum cell area
Min: minimum cell area
Area: Polymegathism distribution
Apex: Pleomorphism distribution
Cell Density
• At birth : 3,000 - 4,000 cell /mm2
• At middle age : 2,500 cell /mm2
• At old age : 2,000 cell /mm2
Endothelial cell loss should be biillaterally symmetrical
Difference > 280 cells/mm2 is clinically significant
Craig T. Use of specular microscopy to
diagnosie corneal disease
Cell Density
• Central endothelial cells density decreases 0.6% / year
• Surgical procedures  the rate of endothelial cell loss
increases 4 times : 2.5% / year for at almost 10 year after surgery
Albon J, Tullo AB, Apoptosis in the endothelium of
human cornea for transplantation. Invest Ophthalmol Vis
Sci 2000; 41: 2887-2893
Coefficient of Variation
• Represents the polymegathism
CV increases with increasing SD
POLYMEGATHISM
Refers to variation in cell sizes
The first sign of endothelial disease
• Normal young adult 0.27
• CV values 0.32 to 0.40 are elevated
• CV values > 0.40 are abnormal
POLYMEGATHISM
CV 45 CD 3268 CV 76 CD 2976 CV 58 CD 3121
POLYMORPHISM
Refers to variation in cell shape
Hexagonal shape <
50% is abnormal
Fuchs Endothelial Dystrophy
Early stage Moderate Severe
ICE Syndrome
ICE cells : large pleomorphic cells with prominent hypereflective nuclei
Liu YK, Wang IJ. Clinical and specular microscopic
manifestation of ICE syndrome. Jpn J Ophthalmol 45. 2001
Dark area with central
spot of light and
peripheral bright zone
THANK YOU
For your kind attention

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Understanding Specular Microscopy.ppt

  • 1. Dr. Johan A. Hutauruk How to read Specular Microscopy
  • 3. Descemet’s membrane Thick basal lamina secreted by endothelium – Anterior banded 3 μm at birth and stable – Posterior non-banded 2 μm at birth, increased to 10 μm in adults • Mainly Type IV collagen, Type VIII and Laminin • Dua’s Layer • Just anterior to Descemet's membare • A very thin and strong layer, 15 microns thick • Function ?
  • 4. Endothelium • Single cell, 4-6 mm thick, total  400,000 cells – At birth :3,500 to 6,000 cell /mm2 – Adult :1,400 to 2,500 cell /mm2 • Hexagonal, 20mm wide : amitotic • Has a single primary cilium : function? • Large nucleus • Smolin G, Thoft RA. The Cornea, 3rd Ed. Boston : Little, Brown & Co., 1994
  • 5. Endothelium • Function : – Effective barrier from the aqueous – Maintain metabolic pump – Maintain corneal transparency
  • 6. Endothelium Kaufman HE, Barron BA, McDonald MB. The Cornea. New York: Churchil Livingstone 1989
  • 7. Endothelium • Cytoplasm : – Numerous mitochondria – Smooth & rough ER – Golgi apparatus • Critical density : Less than 500 cells/mm2 – Not enough room on the lateral cell membrane for the metabolic pump site  corneal edema
  • 8. Endothelium • Endothelial barrier : – Permeable – Tight junction very sensitive to calcium, cause the junction to break down – Also regulated by the cytoskeleton (F-action) of endothelial cells
  • 9. Endothelium • Endothelial pump : – Each cell pumps its own value every 5 minutes – Na+ K+ ATPase • Located in lateral cell membrane • Glucose fueled • 3 million pump sites per endothelial cells – Not fluid pump • Transport ions : Na, bicarbonate from stroma to aqueous • Water follows passively
  • 10. Smolin G, Thoft RA. The Cornea, 3rd Ed. Boston : Little, Brown & Co., 1994
  • 11. Endothelium • Fuchs’ dystrophy : • Endothelial secrete abnormal collagen • Endothelial cells stretch • Permeability increases – Early stages : normal corneal thickness due to increase pump rate – Late stages : corneal edema •Exhausted Na K pump •Lateral cell membrane lack of room for additional pump site
  • 12. VISITING PROFESSORSHIP PROGRAM : INASCRS JEC OPTICAL PRINCIPLE OF SPECULAR MICROSCOPY ▸ Light striking a surface : ▸ Reflected ▸ Transmitted ▸ Absorbed ▸ Specular microscopy : ▸ Light is reflected specularly (mirror-like) ▸ Angle of reflection = angle of incidence
  • 13. INTRODUCTION SPECULAR MICROSCOPE ▸ First described by Maurice (1968) ▸ Modified by Laing, Bourne and Kaufman: replacing the original water immersion lens
  • 15. CORNEAL ENDOTHELIAL CELL MORPHOLOGY • Cell Area ± S.D. (µm2) • Cell Density (cells / mm2) • Polymegethism (CV) • Pleomorphism (% hexagonal 6 sided)
  • 16. Number: the number of analyzed cells CD : cell density AVG : Average cell area SD: standard deviation of cell area CV : Coefficient of variation of cell area Max: maximum cell area Min: minimum cell area Area: Polymegathism distribution Apex: Pleomorphism distribution
  • 17. Cell Density • At birth : 3,000 - 4,000 cell /mm2 • At middle age : 2,500 cell /mm2 • At old age : 2,000 cell /mm2 Endothelial cell loss should be biillaterally symmetrical Difference > 280 cells/mm2 is clinically significant Craig T. Use of specular microscopy to diagnosie corneal disease
  • 18. Cell Density • Central endothelial cells density decreases 0.6% / year • Surgical procedures  the rate of endothelial cell loss increases 4 times : 2.5% / year for at almost 10 year after surgery Albon J, Tullo AB, Apoptosis in the endothelium of human cornea for transplantation. Invest Ophthalmol Vis Sci 2000; 41: 2887-2893
  • 19. Coefficient of Variation • Represents the polymegathism CV increases with increasing SD
  • 20. POLYMEGATHISM Refers to variation in cell sizes The first sign of endothelial disease • Normal young adult 0.27 • CV values 0.32 to 0.40 are elevated • CV values > 0.40 are abnormal
  • 21. POLYMEGATHISM CV 45 CD 3268 CV 76 CD 2976 CV 58 CD 3121
  • 22. POLYMORPHISM Refers to variation in cell shape Hexagonal shape < 50% is abnormal
  • 23. Fuchs Endothelial Dystrophy Early stage Moderate Severe
  • 24. ICE Syndrome ICE cells : large pleomorphic cells with prominent hypereflective nuclei Liu YK, Wang IJ. Clinical and specular microscopic manifestation of ICE syndrome. Jpn J Ophthalmol 45. 2001 Dark area with central spot of light and peripheral bright zone
  • 25. THANK YOU For your kind attention

Editor's Notes

  1. Posterior LImiting Membrane
  2. Permeable because the tight junction do not form complete band (zonula occludens) between endothelial cells. Osmotic gradient draw waters from stroma
  3. David Maurice, Professor of Ocular Physiology at University College, London. Died July 20, 2002
  4. Corneal guttatae