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!!!