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Osmotic Fragility &
RBC Membrane
Defects
Dr Anwar H Siddiqui
Ph.D. Scholar
Department of Physiology, J N Medical College, ...
Meet The Red Cell
 Shaped like a flattened, bilaterally
indented sphere, a biconcave disc
 In fixed, stained blood smear...
Meet The Red Cell
► Durability of Red cell is remarkable
► No nucleus to direct regenerative
processes
► No mitochondria a...
Red Cell Membrane Structure
 Erythrocyte membrane that is normal in structure and
function is essential to survival of re...
Red Cell Membrane Structure
► The red cell membrane consists of:
 Proteins 52%
 Lipids 40%
 Carbohydrates: 8%
 Laminat...
The Red Cell Membrane
Integral Proteins
Lipid Bilayer
Anchoring
Proteins
Cytoskeletal
Proteins
The Membrane Lipids
 Virtually all of the lipids in the mature erythrocyte are
found in the membrane.
Refrence: Wintrobes...
The Membrane Lipids
 The 5 major phospholipids are
asymmetrically disposed, as shown
below:
 Outer monolayer
 Phosphati...
The Membrane Lipids• Macrophages recognize
and phagocytose red cells that
expose PS at their outer surface.
• An exposure ...
Membrane Proteins
Integral proteins
• Embedded in membrane via hydrophobic interactions with lipids.
Peripheral proteins
•...
Integral Proteins
 The red blood cell membrane proteins organized according to
their function:
Transport Proteins Cell Ad...
 Spectrin
 Actin
 Protein 4.1
 Pallidin(band 4.2)
 Ankyrin
 Adducin
 Tropomycin
 Tropomodulin
Peripheral Proteins
...
Peripheral Proteins
Names Definition Function
1. Spectrin
2. Actin
3. Ankyrin
4. Protein 4.1
5. Protein 4.2
cytoskeletal p...
Interactions of RBC Membrane Protein
And Lipids
Disturbed vertical interactions, i.e. disturbed anchoring and membrane
coh...
Interactions of RBC Membrane Protein
And Lipids
Disturbed horizontal interactions
Proteins include: Spectrin , Actin
Membrane Defects
• Hereditary Spherocytosis
Vertical
Interactions
• Hereditary elliptocytosis Syndrome
• Hereditary ellipt...
Hereditary Spherocytosis
 HS is a hemolytic disorder characterized by anemia,
intermittent, jaundice, splenomegaly
 Most...
Hereditary Spherocytosis
ADAR
Defect in 5 possible membrane proteins
Pathophysiology of HS
A) Reduced density of membrane skeleton destabilizes overlying lipid bilayer
B) Loss of Band 3 lipid...
Pathophysiology of HS
Hereditary spherocytosis. A typical Wright-stained peripheral blood smear from a
patient with autosomal dominant hereditar...
Hereditary Elliptocytosis
Syndrome
 The HE syndromes are a family of genetically
determined erythrocyte disorders charact...
 The mechanistic basis for decreased membrane
mechanical stability in HE is weakened “horizontal”
linkages in membrane sk...
CLASSIFICATION OF HEREDITARY
ELLIPTOCYTOSIS SYNDROMES
A B
C
A: Common Hereditary elliptocytosis
B: Hereditary pyropoikilocytosis. Red
cell budding and fragmentation.
C: Southea...
Osmotic Fragility Test
 The osmotic fragility test is a measure
of the ability of the red cells to take up
fluid without ...
The primary factor affecting the osmotic fragility
test is the shape of the red cell, which, in turn,
depends on the
1. V...
 Increased Surface – To – Volume Ratios:
• more resistant to hemolysis and has decreased fragility
• The larger the amoun...
 Decreased Surface – To – Volume Ratios:
• Increased osmotic fragility (decreased
resistance to lysis) is found in
• hemo...
Apparatus And Materials
Tube Method
 Wood or metal test tube rack with 12 clean, dry, 7.5 cm × 1.0 cm
glass test tubes. •...
Procedure (Tube Method)
 Number the test tubes from 1 to 12 with the glass-marking pencil
and put them in the rack.
 Usi...
 Draw 2 ml of blood from a suitable vein and gently eject one
drop of blood into each of the 12 tubes.
 Mix the contents...
Observation and Result
 tube # 1 (normal saline), and tube # 12 (distilled water) will act as
controls, i.e. no hemolysis...
Observation and Result
Express the result in % saline.
 Hemolysis begins in ..... % saline.
 Hemolysis is complete in .....
Procedure (Slide Method)
 Saline solutions of 0.4%, 0.9% and 4.0% strength are prepared.
 One drop of each solution is p...
Normal Range :Initial hemolysis for normal erythrocytes will begin at
0.45 ± 0.05 % NaCl and hemolysis will be complete at...
 When red cells become more fragile, hemolysis may begin at about 0.64%
saline and be complete at about 0.44% saline.
 W...
Plotting the OF graph
 The results of the test may then be graphed, with the percent
hemolysis plotted on the ordinate (v...
 A normal Osmotic Fragility test does not exclude HS
Sensitivity 68% fresh, 81% incubated
Other Tests
 EMA binding test
 Flow cytometry of red cells labeled with eosin-5’-maleimide (EMA)
 HS: sensitivity 93%, ...
Thanks!!!
Osmotic fragility & rbc membrane defects 050916
Osmotic fragility & rbc membrane defects 050916
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Osmotic fragility & rbc membrane defects 050916

  1. 1. Osmotic Fragility & RBC Membrane Defects Dr Anwar H Siddiqui Ph.D. Scholar Department of Physiology, J N Medical College, AMU, Aligarh Physiology Presentation 05-09-2016
  2. 2. Meet The Red Cell  Shaped like a flattened, bilaterally indented sphere, a biconcave disc  In fixed, stained blood smears, erythrocyte appears circular, with a diameter of about 7 to 8 μm and an area of central pallor.  Average values for the mean cellular volume in normal subjects range from 80 to 100 fl.  Highly elastic and deformable. The normal mature erythrocyte as visualized by the scanning electron microscope (×9,800). (Courtesy of Dr. Wallace N. Jensen.) The erythrocyte can pass through a vessel of about 3 μm in maximum diameter
  3. 3. Meet The Red Cell ► Durability of Red cell is remarkable ► No nucleus to direct regenerative processes ► No mitochondria available for efficient oxidative metabolism ► No ribosomes for regeneration of lost or damaged protein ► No de novo synthesis of lipid Images of red blood cells (top) and a human hair (bottom) taken with a confocal microscope Still survives for 120 days!!!
  4. 4. Red Cell Membrane Structure  Erythrocyte membrane that is normal in structure and function is essential to survival of red cell  Accounts for the cell's antigenic characteristics  Maintains stability and normal discoid shape of cell  Preserve cell deformability  Retain selective permeability
  5. 5. Red Cell Membrane Structure ► The red cell membrane consists of:  Proteins 52%  Lipids 40%  Carbohydrates: 8%  Laminated structure consisting of an outer lipid bilayer and a two dimensional network of spectrin-based cytoskeleton.  Cytoskeleton through linking proteins interacts with cytoplasmic domains of membrane proteins.
  6. 6. The Red Cell Membrane Integral Proteins Lipid Bilayer Anchoring Proteins Cytoskeletal Proteins
  7. 7. The Membrane Lipids  Virtually all of the lipids in the mature erythrocyte are found in the membrane. Refrence: Wintrobes Hematology
  8. 8. The Membrane Lipids  The 5 major phospholipids are asymmetrically disposed, as shown below:  Outer monolayer  Phosphatidylcholine (PC);  Sphingomyelin (SM).  Inner monolayer  Phosphatidylethanolamine (PE)  Phosphoinositol (PI).  Phosphatidylserine (PS);
  9. 9. The Membrane Lipids• Macrophages recognize and phagocytose red cells that expose PS at their outer surface. • An exposure of PS can potentiate adhesion of red cells to vascular endothelial cells. • PS can regulate membrane mechanical function, due to their interactions with skeletal proteins such as spectrin and protein 4.1R. The maintenance of an asymmetric phospholipid distribution in the bilayer is critical. Premature destruction of thallassemic and sickle red cells has been linked to disruptions of lipid asymmetry leading to exposure of PS
  10. 10. Membrane Proteins Integral proteins • Embedded in membrane via hydrophobic interactions with lipids. Peripheral proteins • Located on cytoplasmic surface of lipid bilayer, constitute membrane skeleton. • Anchored via integral proteins • Responsible for membrane elasticity and stability.
  11. 11. Integral Proteins  The red blood cell membrane proteins organized according to their function: Transport Proteins Cell Adhesion Protein Structural Proteins AE1- the anion-exchange protein, (formerly knows as Band 3) ICAM-4- interacts with integrins Glycophorins - Imparts a negative charge to the cell, reducing interaction with other cells/ endothelium. •Glycophorin A carry M/N, Gerbich blood group. •Glycophorin C and GlycophorinA, important for P falciparum invasion of RBC. Aquaporin 1 – water transporter, defines the Colton Blood Group Glut1 – glucose and L- dehydroascorbic acid transporter Kidd antigen protein – urea transporter BCAM – a glycoprotein that defines the Lutheran blood groupRhAG – gas transporter defines Rh Blood Group ATPase, co transporter & exchangers
  12. 12.  Spectrin  Actin  Protein 4.1  Pallidin(band 4.2)  Ankyrin  Adducin  Tropomycin  Tropomodulin Peripheral Proteins 4.1
  13. 13. Peripheral Proteins Names Definition Function 1. Spectrin 2. Actin 3. Ankyrin 4. Protein 4.1 5. Protein 4.2 cytoskeletal protein that lines the intracellular side of the plasma membrane. Two subunits: –Alpha and beta, entwined to form dimers. Abundant protein in cell membrane family of adaptor protein is a major structural element. is an ATP-binding protein Responsible for biconcave shape of RBC participates in more protein- protein interactions Interacts with band 3 protein and spectrin to achieve linkage between bilayer and skeleton. Stabilises actin-spectrin interactions. Regulate the association of Band 3 with ankyrin.
  14. 14. Interactions of RBC Membrane Protein And Lipids Disturbed vertical interactions, i.e. disturbed anchoring and membrane cohesion, Proteins include: Ankirin, Band 3 ,Glycoporin and Protein 4.2 etc
  15. 15. Interactions of RBC Membrane Protein And Lipids Disturbed horizontal interactions Proteins include: Spectrin , Actin
  16. 16. Membrane Defects • Hereditary Spherocytosis Vertical Interactions • Hereditary elliptocytosis Syndrome • Hereditary elliptocytosis • Hereditary Pyropoikilocytosis (HPP) • South East Asian Ovalocytosis Horizontal Interactions
  17. 17. Hereditary Spherocytosis  HS is a hemolytic disorder characterized by anemia, intermittent, jaundice, splenomegaly  Most common inherited anemia in Northern European descent  Prevalence 1/1000-2500  75% autosomal dominant fashion  25% Rarely autosomal recessive  Loss of membrane surface area relative to intracellular volume  spherical shape  decreased deformability  splenic destruction
  18. 18. Hereditary Spherocytosis ADAR Defect in 5 possible membrane proteins
  19. 19. Pathophysiology of HS A) Reduced density of membrane skeleton destabilizes overlying lipid bilayer B) Loss of Band 3 lipid-stabilizing effect
  20. 20. Pathophysiology of HS
  21. 21. Hereditary spherocytosis. A typical Wright-stained peripheral blood smear from a patient with autosomal dominant hereditary spherocytosis is shown. Small, dense, round, conditioned spherocytes that lack central pallor are visible throughout
  22. 22. Hereditary Elliptocytosis Syndrome  The HE syndromes are a family of genetically determined erythrocyte disorders characterized by elliptical red cells on the peripheral blood smear.  Inheritance of HE is autosomal dominant (except HPP)  the HE variants occur with an estimated frequency of 1:1,000 to 5,000.  HE has a worldwide distribution, but is more common in malaria endemic regions with prevalence approaching 2% in West Africa.
  23. 23.  The mechanistic basis for decreased membrane mechanical stability in HE is weakened “horizontal” linkages in membrane skeleton due either to defective spectrin dimer-dimer interaction or a defective spectrin- actin-protein 4.1R junctional complex.  The mechanism by which these protein defects result in elliptocyte formation is not clear.
  24. 24. CLASSIFICATION OF HEREDITARY ELLIPTOCYTOSIS SYNDROMES
  25. 25. A B C A: Common Hereditary elliptocytosis B: Hereditary pyropoikilocytosis. Red cell budding and fragmentation. C: Southeast Asian ovalocytosis
  26. 26. Osmotic Fragility Test  The osmotic fragility test is a measure of the ability of the red cells to take up fluid without lysing.  It is a test to measures red blood cell (RBC) resistance to hemolysis when exposed to a series of increasingly dilute saline solutions.
  27. 27. The primary factor affecting the osmotic fragility test is the shape of the red cell, which, in turn, depends on the 1. Volume 2. Surface area 3. Functional state of the red blood cell membrane.
  28. 28.  Increased Surface – To – Volume Ratios: • more resistant to hemolysis and has decreased fragility • The larger the amount of red cell membrane (surface area) in relation to the size of the cell, the more fluid the cell is capable of absorbing before rupturing . As • Example:  Iron-deficiency anemia  Thalassemia  Sickle cell anemia  Liver disease and any condition associated with the presence of target cells
  29. 29.  Decreased Surface – To – Volume Ratios: • Increased osmotic fragility (decreased resistance to lysis) is found in • hemolytic anemias • hereditary spherocytosis • And whenever spherocytes are found
  30. 30. Apparatus And Materials Tube Method  Wood or metal test tube rack with 12 clean, dry, 7.5 cm × 1.0 cm glass test tubes. •Glass marking pencil. •Glass dropper with a rubber teat.  Sterile swabs moist with alcohol. •2 ml syringe with needle.  Freshly prepared 1 percent sodium chloride solution. •Distilled water. Slide Method  Fresh Saline solutions of 0.4%, 0.9% and 4.0% strength.  Sterile swabs moist with alcohol. •2 ml syringe with needle.  Glass slides with cover slips  Microscopes
  31. 31. Procedure (Tube Method)  Number the test tubes from 1 to 12 with the glass-marking pencil and put them in the rack.  Using the glass dropper, place the varying number of drops of 1% saline in each of the 12 test tubes as shown below. Then add the number of drops of distilled water to each of the 12 tubes, as shown
  32. 32.  Draw 2 ml of blood from a suitable vein and gently eject one drop of blood into each of the 12 tubes.  Mix the contents gently by placing a thumb over it.  Leave the test tubes undisturbed for few minutes.  Observe the extent of hemolysis in each tube by holding the rack at eye level, with a white paper sheet behind it.
  33. 33. Observation and Result  tube # 1 (normal saline), and tube # 12 (distilled water) will act as controls, i.e. no hemolysis in normal saline (# 1) and complete hemolysis in distilled water (# 12).  The test tubes in which no hemolysis has occurred, the RBCs will settle down and form a red dot (mass) at the bottom of the tube, leaving the saline above clear.  Some hemolysis, the saline tinged red with Hb, with the unruptured cells forming a red dot at the bottom.  The test tubes with complete hemolysis, the saline will be equally deep red with no red cells at the bottom of these tubes.
  34. 34. Observation and Result Express the result in % saline.  Hemolysis begins in ..... % saline.  Hemolysis is complete in ..... % saline.
  35. 35. Procedure (Slide Method)  Saline solutions of 0.4%, 0.9% and 4.0% strength are prepared.  One drop of each solution is put on three separate slides and one drop of blood is put on each drop of the solutions.  Put a coverslip and observe all the slides for the shape of RBC’s under high power of microscope.
  36. 36. Normal Range :Initial hemolysis for normal erythrocytes will begin at 0.45 ± 0.05 % NaCl and hemolysis will be complete at 0.30 ± 0.05 % NaCl
  37. 37.  When red cells become more fragile, hemolysis may begin at about 0.64% saline and be complete at about 0.44% saline.  When red cells are less fragile, hemolysis starts and is complete at lower strengths of saline.
  38. 38. Plotting the OF graph  The results of the test may then be graphed, with the percent hemolysis plotted on the ordinate (vertical axis) and the sodium chloride concentration on the abscissa (horizontal axis)  Carefully transfer the supernatants to cuvettes and read on a spectrophotometer at a wavelength of 540 nm.  Set the optical density at 0, using the supernatant in test tube #1, which represents the blank, or 0% hemolysis. Test tube #14 represents 100% hemolysis. Calculate the percent hemolysis for each supernatant as follows: Percent of hemolysis = (O.D. of supernatant/ O.D. supernatant tube #14) x 100
  39. 39.  A normal Osmotic Fragility test does not exclude HS Sensitivity 68% fresh, 81% incubated
  40. 40. Other Tests  EMA binding test  Flow cytometry of red cells labeled with eosin-5’-maleimide (EMA)  HS: sensitivity 93%, specificity 99%  Cryohemolysis Increased susceptibility of HS RBCs to rapid cooling (37 to 4°C) in hypertonic solutions  Sensitivity 95%, Specificity 95-96%  Ektacytometry  RBCs suspended in viscous solution as defined values of shear stress applied by ektacytometer, a laser-diffraction viscometer  SDS-Page Electrophoresis  Sodium Dodecyl suphate Polyacrylamide gel electrophoresis  Genetic sequencing
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RBC membrane defects and osmotic fragility test

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