Laboratory Activity 10: Osmotic Fragility Test (Oft)

Laboratory Activity 10:
OSMOTIC FRAGILITY
TEST (OFT)
1
Hereditary Erythrocyte
Membrane Abnormalities
• RBC membrane skeleton

• preserves red cell shape and integrity
2
HEREDITARY SPHEROCYTOSIS
• Blood film
• Microspherocytes
• Pathophysiology
• Spectrin
• Actin
• Protein 4.1
3
• Membrane protein defect

• defective binding of spectrin to protein 4.1
• spectrin deficiency
4
• red cell defect and the splenic

microcirculation
• leads to loss of membrane surface area
• Hemolysis
• membrane permeability is increased
• membrane lipid content is decreased
5
6
• Aplastic crisis
• pancytopenia develops due to lack of
marrow cell production
• Chronic bone marrow hyperplasia
• Skeletal abnormalities
7
• Laboratory Findings and Correlation with
Disease
• Complete Blood Count
• Peripheral Blood Film
• Bone Marrow
• Special Hematology Tests
• Chemistry
8
Complete Blood Count
• Hemoglobin:
• Adults: above 10g/dL
• Infants and young children: lower levels (8-11g/dL)
• MCV:
• 77-87fL
• MCHC:
• increased in ½-2/3 of patients
• Reticulocytes:
• 5%-20%
• Increased in aplastic crisis
• RDW normal to increased
9
Peripheral Blood Film
10
Bone Marrow
11
Chemistry
• slight to moderate rise in unconjugated

bilirubin
• elevated fecal urobilinogen
• decreased haptoglobin levels
12
Special Hematology Tests
• Osmotic Fragility Test

• confirmatory test: increased osmotic fragility
• Direct Antiglobulin Test
• negative in HS
• Autohemolysis test
13
Diagnosis of Hereditary
Spherocytosis
MOST FREQUENTLY • CBC with MCHC
REQUIRED • Peripheral blood film
examination
• Osmotic fragility test
• Reticulocyte count
OCCASIONALLY REQUIRED • Autohemolysis
• Direct antiglobulin test
• Serum unconjugated
(indirect) bilirubin
• Serum haptoglobin
RARELY REQUIRED • Bone marrow examination
14
OSMOTIC FRAGILITY TEST
• reflects the shape of RBCs
• decreased surface area-to-
volume ratio
• spherocytes have a limited
capacity to expand in hypotonic
solutions
• increased osmotic fragility.
• hypochromic or flatter cells
• decreased osmotic fragility.
15
• Specimen requirement
• Fresh heparinized blood
• 15-20 mL of defibrinated whole blood
• Anticoagulants containing oxalates
should NOT be used
• Reagents
• Saline concentrations
• Laboratory made
• Prepackaged
16
• Procedure
• Absorbance:
• Measured spectrophotometrically at 540
17
• Ax%: absorbance in the tube being measured

• A0.85%: absorbance in the 0.85% NaCl tube
• A0.0%: absorbance in the 0.0% NaCl
18
19
20
INCUBATED OSMOTIC
FRAGILITY
• used in mild forms of HS

• whole blood specimen
• incubated for 24 hours at 37C
21
Sources of Error
• Incubated osmotic fragility test

• bacterial contamination
• flasks with beads used to collect
defibrinated blood should be sterilized
• Plasma color is abnormal
• replace plasma with isotonic saline prior to
testing
22
Sources of Error
• Decreased hemoglobin level and poikilocytosis

• causes decreased osmotic fragility
• Highest absorbance reading will occur in a test
solution at or below 0.30% NaCl
• use the highest absorbance reading obtained in
place of the 0.00% reading in the denominator of
the calculation
23
Sources of Error
• Increased osmotic fragility is characteristic of

HS but may also be seen in other acquired
hemolytic anemias that typically have
spherocytes.
24
DIRECT ANTIGLOBULIN TEST
• useful differential diagnosis

• detects antibody on the red cell
surface
• HS has a negative result
25
Treatment of HS
• Splenectomy
• prevents complications such as aplastic
crisis and gallbladder disease
• should be delayed as long as possible
• Splenectomized patients have a normal
life expectancy.
26
GROUP OUTPUT
• ANSWER RESEARCH QUESTIONS

• 1. Describe the spectrophotometric
determination of OFT
• 2. Why is heparin the recommended
anticoagulant for OFT
27

Laboratory Activity 10: Osmotic Fragility Test (Oft)

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Laboratory Activity 10: Osmotic Fragility Test (Oft)

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Laboratory Activity 10:

• RBC membrane skeleton

• Membrane protein defect

• red cell defect and the splenic

• slight to moderate rise in unconjugated

• Osmotic Fragility Test

• Ax%: absorbance in the tube being measured

• used in mild forms of HS

• Incubated osmotic fragility test

• Decreased hemoglobin level and poikilocytosis

• Increased osmotic fragility is characteristic of

• useful differential diagnosis

• ANSWER RESEARCH QUESTIONS

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