RBC membrane defects and osmotic fragility test

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. 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. 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. 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. 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. The Red Cell Membrane
Integral Proteins
Lipid Bilayer
Anchoring
Proteins
Cytoskeletal
Proteins

7. The Membrane Lipids
 Virtually all of the lipids in the mature erythrocyte are
found in the membrane.
Refrence: Wintrobes Hematology

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. 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. 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. 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.  Spectrin
 Actin
 Protein 4.1
 Pallidin(band 4.2)
 Ankyrin
 Adducin
 Tropomycin
 Tropomodulin
Peripheral Proteins
4.1

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. 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. Interactions of RBC Membrane Protein
And Lipids
Disturbed horizontal interactions
Proteins include: Spectrin , Actin

16. Membrane Defects
• Hereditary Spherocytosis
Vertical
Interactions
• Hereditary elliptocytosis Syndrome
• Hereditary elliptocytosis
• Hereditary Pyropoikilocytosis (HPP)
• South East Asian Ovalocytosis
Horizontal
Interactions

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. Hereditary Spherocytosis
ADAR
Defect in 5 possible membrane proteins

19. Pathophysiology of HS
A) Reduced density of membrane skeleton destabilizes overlying lipid bilayer
B) Loss of Band 3 lipid-stabilizing effect

20. Pathophysiology of HS

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

23. 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.

24.  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.

25. CLASSIFICATION OF HEREDITARY
ELLIPTOCYTOSIS SYNDROMES

26. A B
C
A: Common Hereditary elliptocytosis
B: Hereditary pyropoikilocytosis. Red
cell budding and fragmentation.
C: Southeast Asian ovalocytosis

27. 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.

28. 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.

29.  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

30.  Decreased Surface – To – Volume Ratios:
• Increased osmotic fragility (decreased
resistance to lysis) is found in
• hemolytic anemias
• hereditary spherocytosis
• And whenever spherocytes are found

31. 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

32. 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

33.  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.

34. 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.

35. Observation and Result
Express the result in % saline.
 Hemolysis begins in ..... % saline.
 Hemolysis is complete in ..... % saline.

36. 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.

37. 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

38.  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.

39. 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

41.  A normal Osmotic Fragility test does not exclude HS
Sensitivity 68% fresh, 81% incubated

42. 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

43. Thanks!!!