Hema II Chapter 3 - OFT

Chapter three
OSMOTIC FRAGILITY TEST

OFT
 Determines the resistance of the red cell to hemolyis in varying
concentrations of hypotonic solutions.

 Gives an indication of the surface area: volume ratio of
erythrocytes SA:V ratio

 Results of OFT are increased in blood sample ( erythrocytes)
with decrease SA / V ratio
-Useful in the diagnosis & confirmation of hereditary

spherocytosis.
 To maintain viable cells in vivo/vitro the cells must be
suspended in a solution with an osmotic concentration relatively

equal to their interior osmotic concentration: ISOTONIC :0.85%
NaCl solution.
Cont..
 Normal Healthy erythrocytes can tolerate a broad range of
Hypotonic osmotic concentrations (0.85 to 0.55%) due to their

ability to expand or collapse as the environment changes.
This results from RBCs BICONCAVE shape (allows the cell to

increase its volume by about 70%
 When RBCs are put in hypotonic solution the cells take up
water and swell until an equilibrium is attained and then the

cell ruptures
- Normal hemolysis is proceeded by a phase in which the red

cells assume a spherical shape
-Lysis occurs (Na Cl) < 0.55%

Cont..
Spherocytes have:
- a decreased SA / V ratio
- thinner surface membrane
-Lost ability to expand under osmotic pressure
-Take up less water in a hypotonic solution than
normal red cells (<0.7%)
Lyse in less hypotonic ( <0.7%) solution than normal
shaped, biconcave RBCs and therefore have increased
OF ( resistance decreased).
Cont..
Large flat cells such as target cells, have the
ability to take in more water then normal
RBCs, resulting in a decreased osmotic
fragility.
Decreased OF= or increased resistance seen in
Sickle cell anemia
Hypochromic Anemia
IDA , thalassemia,
target cell
Test Procedure
Principle :- Test and normal red cells are placed in a series
of graded strength NaCl solution and resulting
Hemolysis compared a 100% standard.
 Reagents :-
 Stock buffered NaCl solution (10%).
-NaCl (powder) -180g

-Na2 HPO4 27.31g
-NaH2PO4.2H2O 4.86g .......
-Buffering effect
-keep Ph 7.4
 Make up to 2L= D.H2O
 Prepare a1% solution from the stock 10% and proccess as
follows
Cont..
 Procedure
 Mix the contents of each tube (5ml) dilutions:
- NB the 12 dilutions are set up in duplicate (Test &
control)
 Add 0.05ml (50) of pt’d blood to each of the 12 tubes
marked test
 Add the 50l amount (0.05ml) of the normal control blood

in the 2nd row of tubes marked control.
 Mix each tube well
 Let stand at RTO for 30min. Remix & contrifuge at 1000G
for 10 min.
 Separate the supernatants carefully without including
cells.
 Measure Absorbances and of supernatants
Use tube No. 12 of test and control of BLANK
Use tube No 1 of test and control 100% Hemolysis
standard
Cont..
Calculation: Calculate % Hemolysis of each tube
% Hemolysis = Abs T- Abs B X 100
Abs Std – AbsB
But, Abs B (tabe 12) = 0.00
% Hemolysis = Abs T X 100

Abs Std
Cont..
Ex = Abs. Tube- 1 (100% STD) = 0.40
Abs. Tube. 5 = 0.20
% Hemolysis of tube 5 = 0.20 X 100 = 50%
0.40
Cont..
 Reporting of Results: Red cell fragility is best
reported as a curve on a linear graph,

 Inspect the entire fragility curve
-Include the normal control
-Indicate the concentration of Salt in which;
(1) The highest concentration at which Hemolysis

begins
(2) The highest concentration Hemolysis is complete
(3) 50% hemolysis occurred

-Median corpuscular fragility [MCF]
Cont..
 Normal Range (200c, PH 7.4)
Tube No NaCl% Hemolysis (%)
1. 0.10 100%
2. 0.20 100%
3. 0.30 99-
100%
4. 0.35 90-99%
5. 0.40 50-98%
6. 0.45 6-49%
7. 0.50 0.5%
8. 0.55 0%
9. 0.60 0%
10. 0.65 0%
11. 0.70 0%
12. 0.80 0%
Cont..
Saline concentration at which Hemolysis
begins
0.45 – 0.50%
Saline concentration at which 50% RBC
lyse( MCF)
0.40 – 0.45%
Saline concentration at which Hemolysis is
complete
0.30 – 0.35%
Cont..
 FINDINGS
Elevated values high OF= increased V / SA
-Hereditary spherocytosis
-
-Acquired immune Hemolytic -
Anemia with Spherocytesis
-Hereditary stomatocytrsis
Cont..
Decreased Values decreased (V/SA)
=decreased OF
In Hypochromic microcytic cells,
Iron deficency Anemia
Thalassemia
Leptocytosis (target cells)
Cont..
OF after Incubation
 Patients of spherocytosis who are mildly affected
may have fewer than 1% - 2% spherocytes in the

total RBC population.
 It is difficult to diagnose based on morphologic
grounds
 Even they give normal or slight increase OF in
the un-incubated OFT.
Cont..
 Increasing the difference between a normal and
abnormal result is usually possible by increasing
the susceptibility of red cell to osmotic lysis by
prior incubation of the blood at 37oc for 24hrs.
During incubation RBCS (HS cells) become

metabolically deprived and tend to lose
membrane surface because of their relative
less membrane stability.
Both normal subjects & HS patients will have
increased OF after incubation, but the effect
is more marked for patients with HS. In HS
lysis may occur in 8-9 g/L
-set up additional hypotonic solution of 9 g/L
 Also prepare 12 g / L Na Cl solution to use
supernatant as a BLANK
Cont..
Factors Affecting OF
1. Volume of blood & saline

Increase volume blood in 100 volume saline
0.05ml = 1/100
5ml saline
The concentration of blood is so small that the effect of the

PLASMA on the final tonicity of the suspension is negligible.
2.PH of blood in saline suspension
0.1 PH shift is equivalent to altering (saline) by 0.1 g/l a in

PH increase OF
Cont..
3. Temperature
The test is carried out at RTO
Increase TO =decreased OF
50C increase = increase the concentration of
saline by 0.1 g/dl

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Chapter three

OSMOTIC FRAGILITY TEST

concentrations of hypotonic solutions.

erythrocytes SA:V ratio

with decrease SA / V ratio

-Useful in the diagnosis & confirmation of hereditary

suspended in a solution with an osmotic concentration relatively

Hypotonic osmotic concentrations (0.85 to 0.55%) due to their

This results from RBCs BICONCAVE shape (allows the cell to

water and swell until an equilibrium is attained and then the

- Normal hemolysis is proceeded by a phase in which the red

-Lysis occurs (Na Cl) < 0.55%

-NaCl (powder) -180g

 Add the 50l amount (0.05ml) of the normal control blood

But, Abs B (tabe 12) = 0.00

% Hemolysis = Abs T X 100

% Hemolysis of tube 5 = 0.20 X 100 = 50%

reported as a curve on a linear graph,

-Include the normal control

-Indicate the concentration of Salt in which;

(1) The highest concentration at which Hemolysis

(2) The highest concentration Hemolysis is complete

(3) 50% hemolysis occurred

may have fewer than 1% - 2% spherocytes in the

During incubation RBCS (HS cells) become

1. Volume of blood & saline

The concentration of blood is so small that the effect of the

2.PH of blood in saline suspension

0.1 PH shift is equivalent to altering (saline) by 0.1 g/l a in

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