2023-2024

Hematology 2

FINALS
Laboratory Test (part 2)

BACHELOR OF SCIENCE IN MEDICAL TECHNOLOGY

PAROXYSMAL COLD HEMOGLOBIURIA (PCH)  If the serum of the tube incubated in the cold is
 Paroxysmal cold hemoglobinuria (PCH) is a rare hemoglobin-tinged and the serum of the tube that
form of autoimmune hemolytic anemia. remained at 37o C is clear, the patient has a
 It was characteristically associated with syphilis in Donath Landsteiner antibody.
the past, but nowadays is more often associated  If both the cold and warm incubated tubes are
with acute viral infections. hemolyzed, no conclusion can be drawn about the
 PCH can also present as an idiopathic cold presence of a Donath-Landsteiner antibody.
hemagglutinin syndrome.
 The autoantibody that causes this syndrome is DONATH-LANDSTEINER AB
called the Donath Landsteiner antibody. SPECIMEN: Collection by
 It is also referred to as a biphasic cold hemolysin, laboratory staff into 2
because it binds to red blood cells only at cold plain tubes, one at 37°C
temperatures and causes complement mediated and one at 4°C.
hemolysis only after warming to body temperature. METHOD: Supernatant serum
 The autoantibody often has specificity for the P examined for evidence of
blood group antigen. red cell lysis, comparing
a tube incubated at 4°C
How to use Donath-Landsteiner test to diagnose then 37°C with a tube
paroxysmal cold haemoglobinuria (PCH)? maintained at 37°C.
 Paroxysmal cold haemoglobinuria (PCH) accounts REFERENCE No lysis in either tube.
for around a third of cases of autoimmune INTERVAL:
haemolytic anaemia in children. APPLICATION: Diagnosis of paroxysmal
 PCH is caused by an autoantibody that fixes cold haemoglobinuria.
complement to red cells at low temperatures and
dissociates at warmer temperatures (a biphasic INTERPRETATION: The Donath-Landsteiner
haemolysin), triggering complement-mediated antibody is an IgG
intravascular haemolysis. autoantibody that binds
 Named the Donath-Landsteiner (D-L) antibody to red cells in the cold
after its discoverers, it is usually formed in and fixes complement;
response to infection and demonstrates specificity lysis occurs when cells
for the ubiquitous red cell P-antigen. are warmed to 37°C.
 A D-L test can be used to detect the presence of Presence of the antibody
the D-L autoantibody in the patients’ serum. is diagnostic for
paroxysmal cold
 The test is performed by drawing two tubes of haemoglobinuria. This
blood. One is immediately incubated at 37o C for rare disorder may occur
one hour. in syphilis and after viral
 The second tube is immediately incubated in an infections.
ice bath for 30 minutes and then transferred to a
37o C water bath for an additional 30 minutes.
 Both tubes are then centrifuged and examined for
hemolysis.
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OSMOTIC FRAGILITY TEST Clinical Significance
 Elevated values high OFT= increased V / SA
Objectives associated with:
 Define osmotic fragility test  Hereditary spherocytosis
 Describe the procedure of the osmotic fragility test
 Interpret findings of the osmotic fragility test  Acquired immune Hemolytic
 Discuss the purpose of performing an osmotic -
fragility test  Anemia with Spherocytesis
 Correlate hematologic disorders associated with  Hereditary stomatocytrsis
decreased and increased osmotic fragility test  Decreased Values low OFT= decreased V / SA
findings  Associated with abnormal RBC morphology:
o Sickle cells (seen in Sickle cell anemia)
Outline
 Hypochromic microcytic cells(seen in IDA and
 Osmotic fragility definition Thalassemia)
 Osmotic fragility procedure  Leptocytosis (target cells)
 Reporting of OFT results
 Interpretation of osmotic fragility test findings
 Sources of error OFT cont..

Osmotic Fragility Test

 Measures the resistance of RBCs to hemolysis by
osmotic stress
 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  Spherocytes have:
 Useful in the diagnosis & confirmation of hereditary • a decreased SA / V ratio
spherocytosis. • thinner surface membrane
• higher volume (already spherical)
 To maintain viability, the cells must be suspended • Lost ability to expand under osmotic
in a solution with an osmotic concentration pressure
relatively equal to their interior osmotic
concentration: ISOTONIC :0.85% NaCl solution. Test Principle
 Normal healthy erythrocytes can tolerate a broad  Patient sample and normal red cells are placed in
range of Hypotonic osmotic concentrations (0.85 a series of graded strength NaCl solution and
to 0.55%) due to their ability to expand or collapse resulting
as the environment changes.  Hemolysis is compared to a 100% standard.
 This results from RBCs BICONCAVE shape  Reagents :
(allows the cell to increase its volume by about  Stock buffered NaCl solution (10%).
70%  NaCl (powder) = 180g
 Large flat cells such as target cells, have the ability  Na2 HPO4 = 27.31g Disodium phosphate
to take in more water than normal RBCs, resulting  NaH2PO4.2H2O = 4.86g Sodium dihydrogen
in a decreased osmotic fragility. phosphate dihydrate
 Is a screening test for the diagnosis of anemias  Buffering effect
 keep Ph 7.4
 Make up to 2L= d.H2O

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 From (the stock solution, prepare first a 10 g/L
solution by dilution with water. Dilutions equivalent
to 9.0, 7.5, 6.5, 6.0, 5.5, 5.0, 4.0, 3.5, 3.0, 2.0, 1.0
g/L are then prepared.
OFT cont..
 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%
• Two method
• Dacie’s
• Sanford
Osmotic Fragility Test Method
 Heparinized venous blood is used.
 Oxalated or citrated blood is unacceptable due to
additional salts added to it.
 The test should be carried out within 2 hours of
collection if kept at room temperature (15-20O C)
or within 6 hours if kept at 4° C.
Note:
 Procedure requires a minimum of equipment
 The critical point is not that the amount be exactly
50 μl, but rather that the amount added to each
tube must be the same.
 The sigmoid shape of the normal OF curve
indicates that normal red cells vary in their
resistance to hypotonic solutions.
 Indeed, this resistance varies gradually
(osmotically) as a function of red cell age, with the
youngest cells being the most resistant and the
oldest cells being the most fragile. The reason for
this is that old cells have a higher sodium content
and a decreased capacity to pump out sodium.
Figure Osmotic fragility curves. Osmotic fragility curves
of patients suffering from the following: sickle cell anemia,
β-thalassaemia major, hereditary spherocytosis, and
Osmotic Fragility Test Method (Procedure)
A. Deliver 5.0 ml of the 11 saline solutions in test
tubes.
B. Add 5.0 mL of water to tube 12.
C. Add to each tube 50 µL (microlitres) of well mixed
blood and mix immediately by inverting the tubes
for several times avoiding foam.
D. Incubate at room temperature for 30 min.
E. Mix again and centrifuge 1000g for 5 min.
F. Remove the supernatants carefully without
including cells and estimate the amount of lysis in
a photometer at 540 nm.or colorimeter provided
with a yellow-green filter.
G. Use as blank the supernatant from tube 1
(osmotically equivalent to 9 g/l NaCl).
H. Assign a value of 100 % lysis to reading with the
supernatant of tube 12 (water).
OFT Method Cont’d and Calculation
 Express the readings from the other tubes as a
percentage of the value of tube 12
 Plot the results against the NaCl concentration
(Fig.)
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
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“idiopathic” warm autoimmune hemolytic anemia. The OFT Normal Range
normal range is indicated by the unbroken lines (Dacie • Normal Range (200C, PH 7.4)
and Lewis 2006) Tube No NaCl% Hemolysis (%)
1 0.10 100%
Example Calculation 2 0.20 100%
3 0.30 99-100%
Abs. Tube- 1 (100% STD) = 0.40 4 0.35 90-99%
5 0.40 50-98%
Abs. Tube. 5 = 0.20 6 0.45 6-49%
7 0.50 0.5%
% Hemolysis of tube 5 = 0.20 X 100 = 50% 8 0.55 0%
0.40 9 0.60 0%
10 0.65 0%
Interpretation of OFT Results 11 0.70 0%
12 0.80 0%
 Saline concentration at which Hemolysis begins
0.45 – 0.50% OFT after Incubation
 Saline concentration at which 50% RBC lyse  Normal OFT at room temperature does not rule
( MCF) out hereditary spherocytosis because patients
0.40 – 0.45% who are mildly affected may have fewer than 1 -
 Saline concentration at which Hemolysis is 2% spherocytes in the total RBC population.
complete  Diagnose should not be based on morphologic
0.30 – 0.35% grounds
 Therefore, patient blood samples should be
Reporting of Results incubated at 37OC for 24 hours and OFT be
 Report Red cell fragility test results using a curve repeated
on a linear graph as increased, decreased, or  even they give normal or slight increase OFT in
normal the un-incubated OFT.
 Inspect the entire fragility curve
 Include the normal control  Increasing the difference between a normal and
 Indicate the concentration of Salt in which; abnormal result is usually possible by increasing
the susceptibility of red cell to osmotic lysis by
1. The highest concentration at which Hemolysis prior incubation of the blood at 37oc for 24hrs.
begins  During incubation RBCS (HS cells) become
2. The highest concentration Hemolysis is complete metabolically deprived and tend to lose
3. 50% hemolysis occurred membrane surface because of their relative less
 Median corpuscular fragility [MCF] 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 NaCl thus set up
additional hypotonic solution of 9 g/L NaCl
 Also prepare 12 g / L Na Cl solution to use
supernatant as a BLANK

Osmotic Fragility Test Sources of Errors

1. Relative volumes of blood and saline.

2. Final pH of the blood in saline suspension.
3. Temperature at which the tests are carried out.

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 A proportion of 1 volume of blood to 100 volumes
of saline is chosen to render the effect of the
plasma on the final tonicity of the suspension
negligible.
 The fragility of the red cells is increased by a fall in
pH.
 Increase in temperature decreases the fragility, a
rise of 5° C being equivalent to an increase in
saline concentration of about 0.1 g/L

OSMOTIC FRAGILITY TEST VIDEO

https://youtu.be/qhni0TDcYWs?si=oButGAkAKpEPY8B7

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