Osmotic fragility & rbc membrane defects 050916

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Osmotic Fragility & RBC Membrane Defects

Osmotic Fragility & RBC Membrane Defects

Dr Anwar H SiddiquiPh.D. ScholarDepartment of Physiology, J N Medical College, AMU, Aligarh

Physiology Presentation05-09-2016

Meet The Red CellShaped like a flattened, bilaterally indented sphere, a biconcave discIn fixed, stained blood smears, erythrocyte appears circular, with a diameter of about 7 to 8 m 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 m in maximum diameter

Meet The Red CellDurability of Red cell is remarkableNo nucleus to direct regenerative processesNo mitochondria available for efficient oxidative metabolismNo ribosomes for regeneration of lost or damaged proteinNo de novo synthesis of lipid

Images of red blood cells (top) and a human hair (bottom) taken with a confocal microscopeStill survives for 120 days!!!

Red Cell Membrane Structure Erythrocyte membrane that is normal in structure and function is essential to survival of red cellAccounts 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 ProteinsLipid BilayerAnchoring ProteinsCytoskeletal Proteins

The Membrane LipidsVirtually all of the lipids in the mature erythrocyte are found in the membrane.

Refrence: Wintrobes Hematology

The Membrane LipidsThe 5 major phospholipids are asymmetrically disposed, as shown below:Outer monolayerPhosphatidylcholine(PC);Sphingomyelin(SM).Inner monolayerPhosphatidylethanolamine(PE)Phosphoinositol(PI).Phosphatidylserine(PS);

The Membrane LipidsPremature destruction of thallassemic and sickle red cells has been linked to disruptions of lipid asymmetry leading to exposure of PS

Membrane Proteins

Integral ProteinsThe red blood cell membrane proteins organized according to their function:

Transport ProteinsCell Adhesion ProteinStructural ProteinsAE1- the anion-exchange protein, (formerly knows as Band 3)ICAM-4- interacts with integrinsGlycophorins - 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 theColton Blood GroupGlut1 glucose andL-dehydroascorbic acidtransporterKidd antigen protein urea transporterBCAM a glycoprotein that defines the Lutheran blood groupRhAG gas transporter defines Rh Blood GroupATPase, co transporter & exchangers

SpectrinActinProtein 4.1Pallidin(band 4.2)AnkyrinAdducinTropomycinTropomodulin

Peripheral Proteins


Peripheral Proteins



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 proteinResponsible 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 LipidsDisturbed horizontal interactionsProteins include: Spectrin , Actin

Membrane Defects

Hereditary Spherocytosis

HS is a hemolytic disorder characterized by anemia, intermittent, jaundice, splenomegalyMost common inherited anemia in Northern European descent Prevalence 1/1000-250075% autosomal dominant fashion25% Rarely autosomal recessiveLoss of membrane surface area relative to intracellular volume spherical shape decreased deformability splenic destruction

Hereditary Spherocytosis


AR Defect in 5 possible membrane proteins

Pathophysiology of HS

Reduced density of membrane skeleton destabilizes overlying lipid bilayerB) 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 SyndromeThe 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.


ABCA: Common Hereditary elliptocytosis

B: Hereditary pyropoikilocytosis. Red cell budding and fragmentation.

C: Southeast Asian ovalocytosis

Osmotic Fragility TestThe 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 VolumeSurface areaFunctional state of the red blood cell membrane.

Increased Surface To Volume Ratios:more resistant to hemolysis and has decreased fragilityThe 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 anemiaThalassemiaSickle cell anemiaLiver 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 MethodWood 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 MethodFresh 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 slipsMicroscopes

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 Resulttube # 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 ResultExpress 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 RBCs 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 co