Erythrocyte Membrane Fragility

dr. Lusiana Batubara, M.Si.Med

 Red Blood Cell

• RBC are small cell (6-8 μm in diameter)

• Transport O2 from lungs to the peripheral tissues.
• Disposal of CO2and [H+] protons formed during
tissue metabolism.
• RBCs must be able to squeeze through some tight
spots in micro-circulation (about 3 μm) without
fragmentation  for that RBCs must be easily &
reversibly deformable.
 Red Blood Cell

• RBC are not true cells.

• Composed of Membrane surrounding a solution of
hemoglobin (95%).
• Bag filled with hemoglobin.
 Red Blood Cell

• RBC contain no nucleus or nucleic acids

• RBC contain no cell organelles (as mitochondria,
Golgi, ER or lysosomes)
• RBC contain no mitochondria, so there is no
respiratory chain, no citric acid cycle, and no
oxidation of fatty acids or ketone bodies.
 Red Blood Cell

• RBC contain no nucleus or nucleic acids

• RBC contain no cell organelles (as mitochondria,
Golgi, ER or lysosomes)
• RBC contain no mitochondria, so there is no
respiratory chain, no citric acid cycle, and no
oxidation of fatty acids or ketone bodies.
 RBC Metabolism

• RBC is highly dependent upon glucose as its energy

source.
• Energy in the form of ATP is obtained ONLY from the
glycolytic breakdown of glucose with the production
of lactate (anaerobic glycolysis).
 RBC Metabolism

• Glucose is metabolized in RBCs through anaerobic

glycolysis (that requires no mitochondria and no
oxygen)
• One molecule of glucose yields 2 molecules of ATP
• In addition, 2 molecules of lactate are produced.
• Lactateis transported to blood & in the liver it is
converted to glucose.
 RBC Metabolism

Importance of glycolysis in red cells:

• Energy production: it is the only pathway that supplies the red
cells with ATP.
• Reduction of methemoglobin: glycolysis provides NADH for
reduction of metHb by NADH-cytochrome b5 reductase
• Reduction of Glutathione: glycolysis provides NADH for
reduction of Glutathione. Reduced glutathione is necessary
for the integrity of the erythrocyte membrane (prevents
oxidation of membrane)
• Generate 2,3 bisphosphoglycerate (2,3-BPG). 2,3-BPG binds to
Hb, decreasing its affinity for O2, and helps its availability to
tissues.
 Reduction of Glutathione

Generate 2,3-BPG

Reduction of methemoglobin

Energy production
 RBC Membrane

• Under the microscope, the

red blood cell appears to be a
red disc with a pale central
area (biconcave disc)
• The biconcave disc shape
serves to facilitate gas
exchange across the cell
membrane.
 RBC Membrane

• The membrane proteins that

maintain the shape of the red
blood cell also allow the red blood
cell to traverse the capillaries with
very small luminal diameters to
deliver oxygen to the tissues.
• The interior diameters of many
capillaries are smaller than the
approximately 8μm diameter of
the red cell.
 RBC Membrane

• About 50% of membrane is protein, 40% is fat & up to 10% is

carbohydrate.
• RBC membrane comprises a lipid, proteins that are either
peripheral or integral penetrating the lipid bilayer &
carbohydrates that occur only on the external surface.
 RBC Membrane

• The major lipid classes in membrane are phospholipids and

cholesterol

• Major Phospholipids are :

Phosphatidylcholine (PC) The choline-containing phospholipids, predominate
Sphingomyelin (Sph) in the outer leaflet

Phosphatidylethanolamine (PE) The amino-containing phospholipids

predominate in the inner leaflet.
Phosphatidylserine (PS)
Glycosphingolipids (GSLs) 5–10%
ABO blood group substances
 RBC Membrane

The major membrane Proteins

• The membrane skeleton is four structural proteins that
include α & β spectrin, ankyrin, protein 4.1 & actin.
• Spectrin is major protein of the cytoskeleton
• Defects of proteins may explain some of the abnormalities of
shape of RBC membrane as
 hereditary spherocytosis
Haemolytic anemia
 elliptocytosis
Osmotic Fragility Test
 Osmotic Fragility Test

• RBC are exposed to a series of saline (NaCl) solutions with

increasing dilution
• The sooner hemolysis occurs, the greater is osmotic fragility
of RBC

% NaCl % Lisis
0,20 97-100
0,30 90-98
0,35 50-95
0,40 5-45
0,45 0-6
 Osmotic Fragility Test

Factors affecting osmotic fragility:

• cell membrane permeability
• Surface area-to-volume ratio

Increased osmotic fragility

• Hereditary spherocytosis
• Acquired spherocytosis

Decreased osmotic fragility

• Thalassemia
• Sickle cell anemia
 Osmotic Fragility Test

Alat dan Bahan:

• larutan NaCl dengan konsentrasi 0%; 0,20%; 0,30%; 0,35%;
0,40%; 0,45%; 0,50%; 0,90%
• Darah EDTA
• Spektrofotometer Stat Fax 3300
• Micropipet 50 µl
• 8 buah tabung sentrifuse
• Injeksi spuit
 Osmotic Fragility Test

Cara Kerja:
1. Siapkan 8 tabung sentrifuge, berilah tanda pada masing-masing
tabung dengan angka: 0, 0,20; 0,30; 0,35; 0,40; 0,45; 0,50; 0,90
2. Masukkan 10 ml larutan NaCl dengan konsentrasi yang berbeda-
beda ke dalam masing-masing tabung yang telah diberi tanda
3. Tambahkan 0,1 ml darah EDTA pada masing-masing tabung dan
campur dengan baik sampai darah merata dalam larutan NaCl
4. Diamkan pada temperatur kamar selama 60 menit dan kemudian
sentrifuse pada 3000 rpm selama 5 menit
5. Ambil 3 ml supernatan dan lakukan pembacaan dengan
menggunakan spektrofotometer pada 540 nm. Pembacaan
blangko adalah dari supernatan 0,9%.
 Osmotic Fragility Test

• Untuk menghitung berapa banyak hemolisis yang terjadi

pada masing-masing tabung, maka pembacaan masing-
masing tabung dibandingkan dengan pembacaan OD
tabung 0

• % hemolisis = OD sampel x 100

OD tab 0
 Osmotic Fragility Test

Interpretasi:
Menggunakan data berikut

% NaCl % Lisis
0,20 97-100
0,30 90-98
0,35 50-95
0,40 5-45
0,45 0-6
 Osmotic Fragility Test

Interpretasi:
1. Buatlah grafik standard tersebut di atas dan buatlah grafik
hasil percobaan kelompok saudara
2. Bandingkan dengan grafik standart, maka saudara akan
dapat memperoleh interpretasi hasil kerja saudara.
Thank you