OSMOTIC FRAGILITY TEST

The osmotic fragility test compares the resistance of normal and pathologic red cells to various
osmotic pressures. Red cells suspended in hypotonic solutions take up water, swell, become spheroid,
and burst after reaching a critical volume.
The chief value of the OF test is for diagnosing Hereditary Spherocytosis. Measures how
spheroid red cells are (not how fragile), i.e., estimates the surface-to-volume ratio of red cells.

Objectives:
1. Explain and discuss the principle of OFT;
2. Determine the acceptable specimen for Osmotic Fragility testing;
3. Compute the % Hemolysis of the test specimen using a spectrophotometer or colorimeter and
4. Plot the % Hemolysis in a graph and interpret the OFT result.
Procedure:
1. Fill each tube with the correct volume of NaCl solution and water. Follow the table below.

Tube NaCl (%) Amount of NaCl (mL) Amount of Distilled water (mL)
1 0.00 0 5
2 0.25 1.25 3.75
3 0.35 1.75 3.25
4 0.40 2.0 3.0
5 0.45 2.25 2.75
6 0.50 2.5 2.5
7 0.55 2.75 2.25
8 0.60 3.0 2.0
9 0.65 3.25 1.75
10 0.70 3.5 1.5
11 0.75 3.75 1.25
12 0.85 4.25 0.75

2. Add 50 µL of well-mixed heparinized blood to each tube and mix immediately by inverting the lines
several times to avoid foam. Add to each tube 50 µL of well-mixed heparinized blood and mix
immediately by inverting the tubes several times to avoid foam.
3. Incubate at room temperature for 30 min. Mix again and centrifuge 1000g for 5 min.
4. 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.
5. Calculate the % Hemolysis of each tube. tube.

Formula: Absorbance of individual test tube x 100

The absorbance of 100% hemolysis

6. Plot the results against the NaCl concentration.

Note: Use tube 1 for 100% Hemolysis

Sample graph

Control Control
Test

Test
 Table 1

Osmotic Fragility Test Results

Tube NaCl (%) Absorbance % Hemolysis
1 0.00 0.196 100.00%
2 0.25 0.200 102.04%
3 0.35 0.062 31.63%
4 0.40 0.205 104.59%
5 0.45 0.206 105.10%
6 0.50 0.138 70.41%
7 0.55 0.102 52.04%
8 0.60 0.203 103.57%
9 0.65 0.219 111.73%
10 0.70 0.195 99.49%
11 0.75 0.193 98.47%
12 0.85 0.024 12.24%

Figure 2

Osmotic Fragility Curve

120%

100%

80%
Hemolysis (%)

60%

40%

20%

0%
0 1 2 3 4 5 6 7 8
0. 0. 0. 0. 0. 0. 0. 0.
NaCl Concentration (%)

Upon reviewing the laboratory results for the osmotic fragility test, significant procedural errors
are evident. The data do not exhibit the expected logarithmic inverse relationship or the characteristic
sharp bend curve typically associated with such hemolysis experiments. Notably, the percentage of
hemolysis in several tubes exceeds 100% (e.g., Tubes 2, 4, 5, 8, and 9), which is physiologically
implausible and indicates erroneous readings. Additionally, the hemolysis percentages do not
decrease consistently with increasing NaCl concentration, as would be anticipated in a properly
conducted osmotic fragility test. The discrepancies observed, such as the unexpectedly high
hemolysis values at higher NaCl concentrations and the lack of a clear trend, strongly suggest issues
in the experimental procedure rather than in the instrument calibration or quality control. These
anomalies could be attributed to factors like improper sample handling, inaccuracies in reagent
preparation, or errors in measurement technique.
Figure 3 Figure 4

Preparation of NaCl solutions Addition of blood sample

Figure 6
Figure 5
Centrifugation
Incubation

Figure 7 Figure 8

1st part of Results 2nd part of Results

 I. Discussion (not more than 1 page)

Gao and Monaghan (2018) explained that the Osmotic Fragility Test (OFT) is based on the
principle of determining how well red blood cells can tolerate hypotonic conditions. Specifically, this
test determines how easily red blood cells may lyse in progressively less osmotic liquids. Because of
their biconcave form, normal red blood cells can swell by around 70% before their surface membrane
breaks down to the point where cell lysis occurs (Layton & Roper, 2017). Spherocytes, which have a
lower surface-to-volume ratio than regular cells, are more likely than normal cells to undergo
premature lysis in hypo-osmotic solutions because of their restricted ability to hold water without
straining their membranes. Increased osmotic fragility is seen in conditions such as hereditary
spherocytosis (HS), which is characterized by a characteristic 'tail' of extremely fragile cells on the
fragility curve. On the other hand, diseases such as thalassemia and iron deficiency anemia, in which
red blood cells have lower volume-to-surface area ratios, show less osmotic fragility. By distinguishing
between spherocytes and normal discoid red blood cells based on their reactions to hypotonic
settings, OFT is an invaluable diagnostic tool for hereditary spherocytic hemolytic anemia and a
variety of other hemolytic diseases.

According to Khalid (2020), complete blood is the appropriate specimen for Osmotic Fragility
testing; this is usually drawn in a lavender-topped tube that contains K2 EDTA anticoagulant. For
reliable testing, a total amount of 6 mL is preferred; however, a minimum volume of 2 mL is advised. A
standard venipuncture is used to get the blood. The collected specimen must be fresh and given to the
laboratory for processing within 72 hours, preferably within 24 hours of the time of collection, in order
to guarantee the validity and trustworthiness of the results.

The calculation of % Hemolysis of the test specimen using a spectrophotometer or colorimeter

involves measuring the optical density of NaCl solutions containing red blood cells (at varying NaCl
concentrations) using a cuvette with a 10 mm thickness, typically within the wavelength range of 670-
750 nm (Tsirkin et al., 2011). The formula used to calculate the percentage of hemolysis in each tube
is 100 × (Dmax - Dx) / (Dmax - Dmin), where Dx represents the test sample's optical density and
Dmax and Dmin stand for the maximum and minimum optical densities, respectively. The calculation
uses the maximum and minimum optical densities as reference points. Dmax is the optical density
obtained with 0.1% NaCl solution, which represents 100% hemolysis, and Dmin is the optical density
obtained with 0.9% NaCl solution, which represents zero hemolysis.

Plotting the percentage of hemolysis on a graph for an osmotic fragility test (OFT) entails
illustrating the link between the amount of hemolysis seen in red blood cells (RBCs) and the saline
solution's concentration (hypotonicity) (Kumar, 2022). When interpreting the osmotic fragility test
(OFT) result graph, a shift to the left for leptocytes implies greater resistance to osmotic challenges
compared to normal RBCs, while a shift to the right for spherocytes implies higher hemolysis at higher
saline concentrations, signifying increased fragility according to Kumar's analogy. This graphical
illustration highlights higher or lower rates of hemolysis connected with differences in the cells' surface
area-to-volume ratios and shows how variations in RBC shapes impact their sensitivity to osmotic
stress.
II. Guide questions

1. What is an acceptable patient and control specimen? Why?

In the Osmotic Fragility Test, patient specimens help identify deviations from the normal
range of osmotic fragility (Ezzelle, Rodriguez-Chavez, et al. 2007). An acceptable patient
specimen is typically a whole blood sample obtained from individuals suspected of having
disorders that may impact red blood cell membrane integrity. These conditions can lead to
increased osmotic fragility due to abnormalities in the RBC membrane or alterations in
hemoglobin content. On the other hand, acceptable control specimens are sourced from
healthy individuals with normal RBC function. These samples are crucial for establishing a
baseline of normal osmotic fragility, which allows accurate comparisons with patient samples. It
is used to evaluate whether a patient's result has been compromised by handling conditions
such as temperature, motion, or other transportation interferences. Moreover, this approach
ensures that observed variations in osmotic fragility can be reliably attributed to the presence
of underlying hematological conditions. Furthermore, testing patient specimens allows
healthcare professionals to evaluate how RBCs from these individuals respond to varying
concentrations of saline solutions, providing valuable information for the diagnosis and
monitoring of specific hematological disorders.

2. Interpret the following OF results:

Control: Patient
Initial 0.50% NaCl Initial 0.30% NaCl
Complete 0.30%NaCl Complete0.20%NaCl

In the control specimen, the erythrocytes started to lyse or undergo hemolysis at 0.50%
NaCl. The Osmotic Fragility (OF) test results for the patient are invalid due to the inappropriate
control used. In OF testing, it's crucial to have a control that reflects the normal range of
erythrocyte osmotic fragility (Pathlabs, 2017). In this instance, the initial NaCl concentration
employed as the control was 0.50%, a far lower quantity than the 0.85% typical control. The
control's validity is jeopardized by this departure from the normative value, rendering it useless
for reliably evaluating the patient's outcomes. A control with a 0.85% NaCl content is often
anticipated to show very little hemolysis in an OF test, acting as a standard for normal
erythrocyte fragility. A falsely high amount of hemolysis may result from using a 0.50% NaCl
solution in the control, which would not correctly reflect the typical erythrocyte reaction to
osmotic stress. As a consequence, it is not possible to consistently evaluate the patient's
findings against this defective control, which begin at an initial NaCl concentration of 0.30%
and demonstrate full hemolysis at 0.20%.

Thus, it is recommended that the OF test be repeated using a control with the correct
0.85% NaCl concentration to ensure accurate and clinically meaningful results.

3. Give an example of one erythrocyte disorder or condition in which OF

results typically fall within the normal range.

Red blood cell (RBC) concentration looks high in relative erythrocytosis, an erythrocyte
disease caused by inadequate plasma (Pandey, 2020). Dehydration is a typical reason since
the body doesn't have enough fluids, which makes the RBCs appear erroneously increased.
The osmotic fragility test determines if RBCs in varying saline solution concentrations will
hemolyze. Even though the concentration of red blood cells appears to be high in relative
erythrocytosis, the results of osmotic fragility usually stay within the normal range. Making the
distinction between true erythrocytosis and relative erythrocytosis is crucial for determining the
 best course of treatment. The osmotic fragility test indicates an RBC's vulnerability to
hemolysis under circumstances, not its quantity.
4. In mild forms of hereditary spherocytosis (HS), the osmotic fragility test
is normal. What can be done to detect these mild cases of HS

In mild versions, hereditary spherocytosis (HS) is an inherited illness that can be

challenging to diagnose (Cleveland Clinic medical professional, 2022). Anemia, jaundice, and
splenomegaly are among the symptoms. Oftentimes, a family history of HS is necessary. Tests
performed in laboratories include bilirubin levels, full blood counts, and blood smears. The
osmotic fragility test evaluates the response of red blood cells to osmotic pressure variations.
One way to distinguish HS from other hemolytic anemias is by a negative Coombs test result.
Genes linked to HS can have mutations revealed through genetic testing; if a genetic mutation
is discovered, family screening is advised. Clinical suspicion and follow-up are crucial. If
symptoms appear or get worse, repeat testing and periodic monitoring may be required.
Accurate diagnosis and treatment depend heavily on close cooperation between
hematologists, geneticists, and healthcare professionals. Hs can vary in severity, and
abnormalities in mild cases may not always be present.

III. Conclusion

In the laboratory, we learned how to perform the OFT test in Hematology. The objectives
were sufficiently achieved as we discussed the principle of OFT, determining the appropriate
specimen, computing the percentage hemolysis, and lastly, how to plot a graph and interpret
the results. The Osmotic Fragility Test is a diagnostic tool used to identify deviations from the
normal range of osmotic fragility. It is used to assess the integrity of red blood cells (RBCs) in
individuals suspected of having disorders that may impact their membranes. Acceptable
patient and control specimens are obtained from healthy individuals with normal RBC function,
allowing accurate comparisons and assessing the presence of underlying hematological
conditions. The test also helps healthcare professionals evaluate how RBCs respond to
varying concentrations of saline solutions, providing valuable information for the diagnosis and
monitoring of specific hematological disorders. An example of an erythrocyte disorder where
OF results typically fall within the normal range is relative erythrocytosis, an erythrocyte
disease caused by inadequate plasma. In mild forms of hereditary spherocytosis (HS), the
osmotic fragility test is normal, but genetic testing can reveal mutations linked to HS. Thus, it is
vital to master the osmotic fragility test—as the fragility of red blood cells might also indicate
the fragility of the patient’s health.
 IV. References

Cleveland Clinic medical professional. (2022, May 19). Hereditary spherocytosis: What it is,
symptoms, causes & treatment. Cleveland Clinic.
https://my.clevelandclinic.org/health/diseases/23058-hereditary-spherocytosis?
fbclid=IwAR0BpmzSvOQulGqQKvNHJdgM5RjZAmpHC70YxKjvGxVxSymc8T8fUsU5XMU

Ezelle, J., Rodriguez-Chavez, I.R., et. Al (2007). Guidelines on Good Clinical Practice. Retrieved from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2213906/

Gao, J., & Monaghan, S. A. (2018). Red blood cell/hemoglobin disorders. In Hematopathology (pp. 3-
56). Elsevier.

Layton, M., & Roper, D. (2017). 12—Investigation of the Hereditary Haemolytic Anaemias: Membrane
and Enzyme Abnormalities. Dacie and Lewis Practical Haematology, 12th ed.; Bain, BJ, Bates,
I., Laffan, MA, Eds, 228-253.

Pandey, A. (2020, January 23). Osmotic fragility test: Procedure, purpose, results, normal range, cost,
Price, online booking. myUpchar. https://www.myupchar.com/en/test/osmotic-fragility?
fbclid=IwAR12SU_v54jHGToSv4RuDFFQuMkuyWBxSOcJDBtcTo3gxg-P4-_H7RHjLtw

Pathlabs (2017). Osmotic Fragility Test — Its Usage, Procedure and Test Result Interpretation.
Retrieved from https://medium.com/@lalpathlabssmo/osmotic-fragility-test-its-usage-procedure-
and-test-result-interpretation-a068b6665da3

Khalid, U. (2020, September 30). Osmotic fragility of erythrocytes. Reference Range, Interpretation,
Collection, and Panels. https://emedicine.medscape.com/article/2085814-overview#a1

Kumar, S. (2002). An analogy for explaining erythrocyte fragility: concepts made easy. Advances in
Physiology Education, 26(2), 134-135. https://doi.org/10.1152/advan.00008.2002

Tsirkin, V. I., Krysova, A. V., & Kunshin, A. A. (2011). Method for evaluating osmotic fragility (Patent
No. RU2419792C1). Russia.