Osmotic Fragility Test (OFT) in Hematology
Sure! Here’s a detailed lecture on the OFT test in hematology (Osmotic Fragility Test), covering its principles, indications, procedure, interpretation, and related topics.
This can be useful for medical students, residents, lab technologists, or anyone studying hematology.
📚 Lecture: Osmotic Fragility Test (OFT) in Hematology
✏️ Outline
Introduction
Principle of the OFT
Types of Osmotic Fragility Tests
Indications and Clinical Applications
Methodology
Classic method (acidified glycerol lysis test)
Modified methods
Factors affecting OFT
Interpretation of results
Increased fragility
Decreased fragility
Limitations
Advances and alternatives
Summary
🧬 1. Introduction
The Osmotic Fragility Test (OFT) is a laboratory test that measures the resistance of red blood cells (RBCs) to hemolysis when exposed to hypotonic saline solutions.
RBCs normally swell in hypotonic solutions due to water influx.
At a critical point, the cell membrane ruptures, causing hemolysis.
The test helps to detect conditions where the RBC membrane is abnormally fragile (e.g., hereditary spherocytosis) or abnormally resistant (e.g., thalassemia).
🔬 2. Principle of the OFT
The test is based on the osmotic behavior of RBCs:
RBCs are suspended in a series of saline solutions of decreasing NaCl concentration.
As the osmotic pressure decreases, water enters RBCs, leading to swelling.
Cells with less surface area relative to volume (e.g., spherocytes) rupture earlier.
Cells with more membrane reserve (e.g., target cells) withstand lower saline concentrations.
The percentage of hemolysis is plotted against the NaCl concentration to get a fragility curve.
🧪 3. Types of Osmotic Fragility Tests
A. Classic Osmotic Fragility Test
Gradual decrease in NaCl concentrations (usually from 0.85% to 0.10%).
B. Acidified Glycerol Lysis Test (AGLT)
Uses glycerol to speed up the test.
C. Pink Test / NESTROFT
Qualitative, rapid screening for thalassemia.
📋 4. Indications and Clinical Applications
Diagnosis of hereditary spherocytosis (HS)
Supportive test in thalassemia trait or disease
Rarely in other membrane disorders: elliptocytosis, stomatocytosis
To evaluate acquired RBC membrane defects
Not a standalone test; used with clinical and other hematological findings.
⚗️ 5. Methodology
Classic Osmotic Fragility Test
Materials
Fresh EDTA blood (<2 hrs old)
Series of NaCl solutions (0.85% → 0.10%)
Phosphate buffer (pH 7.4)
Centrifuge, spectrophotometer
Steps
Mix blood with each saline concentration.
Incubate at room temperature (typically 30 mins).
Centrifuge and measure supernatant absorbance at 540 nm.
Calculate % hemolysis:
% hemolysis=Absorbance of test solution−Absorbance of 0% hemolysisAbsorbance of 100% hemolysis−Absorbance of 0% hemolysis×100\% \text{ hemolysis} = \frac{\text{Absorbance of test solution} – \text{Absorbance of 0\% hemolysis}}{\text{Absorbance of 100\% hemolysis} – \text{Absorbance of 0\% hemolysis}} \times 100
Plot the osmotic fragility curve.
Key values
Initial hemolysis: starts at ~0.45–0.50% NaCl
Complete hemolysis: at ~0.30–0.33% NaCl
Acidified Glycerol Lysis Test (AGLT)
More sensitive for HS
Blood mixed in glycerol-buffered solution; lysis time measured
Faster and uses fewer reagents
Pink Test / NESTROFT
Naked eye single tube red cell osmotic fragility test
Qualitative, low-cost screening for β-thalassemia trait
Tube with 0.36% buffered saline; presence or absence of turbidity indicates fragility
⚠️ 6. Factors Affecting OFT
Age of RBCs: older cells → more fragile
Storage: testing must be done promptly
pH and temperature: must be standardized
Hematocrit and sample volume
Technical handling and pipetting accuracy
📈 7. Interpretation of Results
| Condition | Initial Hemolysis | Complete Hemolysis | Fragility |
|---|---|---|---|
| Normal RBCs | ~0.45–0.50% NaCl | ~0.30–0.33% NaCl | Normal |
| Hereditary spherocytosis | ~0.60–0.65% NaCl | ~0.45% NaCl | Increased |
| Thalassemia, target cells | ~0.35% NaCl | ~0.20–0.25% NaCl | Decreased |
Increased osmotic fragility:
Hereditary spherocytosis
Immune hemolytic anemias
Severe burns
Decreased osmotic fragility:
Thalassemia
Iron deficiency anemia
Liver disease
Post-splenectomy states
🧪 8. Limitations
Not highly specific; other conditions may alter fragility.
Sensitivity depends on method used.
False negatives/positives possible.
Modern molecular and flow cytometry tests are more accurate.
🧬 9. Advances and Alternatives
Eosin-5′-maleimide (EMA) binding test: Flow cytometry for HS (higher sensitivity and specificity).
Molecular testing for membrane protein gene mutations.
More automated osmotic gradient ektacytometry.
✅ 10. Summary
The OFT assesses RBC resistance to osmotic lysis.
It is based on the relationship of cell volume and surface area.
Most useful in diagnosing hereditary spherocytosis and screening thalassemia.
Newer methods are gradually replacing the classic OFT due to higher sensitivity and automation.
📚 References
Dacie & Lewis: Practical Haematology
Bain BJ, Bates I, Laffan MA: Dacie and Lewis Practical Haematology (12th Edition)
Hoffbrand’s Essential Haematology
Recent articles on EMA binding test and osmotic ektacytometry
If you’d like, I can also prepare:
✅ PowerPoint slides
✅ A handout summary
✅ A comparison table of OFT vs EMA binding test
Would you like any of these?
