Osmotic fragility & rbc membrane defects 050916
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Transcript of Osmotic fragility & rbc membrane defects 050916
Osmotic Fragility & RBC Membrane Defects
Dr Anwar H SiddiquiPh.D. Scholar
Department of Physiology, J N Medical College, AMU, Aligarh
Physiology Presentation05-09-2016
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
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!!!
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
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.
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 Hematology
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);
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
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.
Integral Proteins The red blood cell membrane proteins organized
according to their function:Transport Proteins Cell Adhesion
ProteinStructural 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 GroupGlut1 – glucose and L-dehydroascorbic acid transporterKidd antigen protein – urea transporter
BCAM – a glycoprotein that defines the Lutheran blood group
RhAG – gas transporter defines Rh Blood Group
ATPase, co transporter & exchangers
Spectrin Actin Protein 4.1 Pallidin(band 4.2) Ankyrin Adducin Tropomycin Tropomodulin
Peripheral Proteins
4.1
Peripheral ProteinsNames 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.
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
Interactions of RBC Membrane Protein And Lipids
Disturbed horizontal interactionsProteins include: Spectrin , Actin
Membrane Defects
• Hereditary SpherocytosisVertical
Interactions
• Hereditary elliptocytosis Syndrome• Hereditary elliptocytosis• Hereditary
Pyropoikilocytosis (HPP)• South East Asian
Ovalocytosis
Horizontal Interaction
s
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
Hereditary SpherocytosisAD AR
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-stabilizing effect
Pathophysiology of HS
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
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.
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.
CLASSIFICATION OF HEREDITARY ELLIPTOCYTOSIS SYNDROMES
A B
CA: Common Hereditary elliptocytosis
B: Hereditary pyropoikilocytosis. Red cell budding and fragmentation.
C: Southeast Asian ovalocytosis
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.
The primary factor affecting the osmotic fragility test is the shape of the red cell, which, in turn, depends on the 1.Volume2.Surface area3.Functional state of the red blood cell membrane.
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
Decreased Surface – To – Volume Ratios:• Increased osmotic fragility (decreased
resistance to lysis) is found in
• hemolytic anemias
• hereditary spherocytosis
• And whenever spherocytes are found
Apparatus And MaterialsTube 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
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
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.
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.
Observation and Result
Express the result in % saline. Hemolysis begins in ..... % saline. Hemolysis is complete in ..... % saline.
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.
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
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.
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
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%, 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
Thanks!!!