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Red Blood Cells (RBCs), also known as erythrocytes, are the most abundant type of blood cell in the blood. In vertebrates, RBCs transport oxygen throughout the body and also possess immune functions. In anemia testing, defects in the red blood cell membrane can be detected through the red blood cell osmotic fragility test.
Test Principle:
When suspended in an isotonic saline solution, red blood cells maintain their biconcave disc shape. If the osmotic pressure increases, water will flow out of the cells, causing them to shrink. Conversely, if the osmotic pressure decreases, water will enter the cells, causing them to swell and eventually rupture, leading to hemolysis. Based on this principle, red blood cells are added to a series of low‑osmotic saline solutions of different concentrations, and the degree of hemolysis is examined to determine the resistance of the red blood cells to hypotonic solutions. This test is referred to as the red blood cell osmotic fragility test and is primarily used to assess the osmotic fragility of human and animal blood cells. This kit is intended for research use only and is not suitable for clinical diagnosis or other purposes.
Reagents, consumables and Equipments not provided
Small test tubes, spectrophotometer
Procedure
1. Preparation of Parpart NaCl Working Solution
Prepare an appropriate amount of Parpart NaCl working solution by mixing Parpart NaCl Solution and ddH₂O at a ratio of 1:9.
2. Sample Preparation
Collect blood aseptically and prepare heparin‑anticoagulated blood or defibrinated blood. Mix thoroughly before use.
3. Dilution Series
Take small test tubes or appropriate containers, label them, and sequentially add Parpart NaCl working solution and ddH₂O according to the Parpart NaCl dilution table. The total volume per tube should be 4 mL. A normal control should be included in each assay.
4. Adding Blood Samples
Add 40 μL of heparin‑anticoagulated blood or defibrinated blood to each of the above Parpart NaCl dilution tubes. Mix immediately and let stand at room temperature (20–30 °C) for 30 minutes.
5. Measurement
Centrifuge each tube for 5 minutes and take the supernatant. Measure the absorbance at 540 nm or using a green filter on a spectrophotometer. Use the supernatant from the tube containing 8.5 g/L NaCl (tube No. 3) as the blank to zero the instrument. Use the tube containing 1 g/L NaCl (tube No. 17, or a tube in which ddH₂O replaces the Parpart NaCl dilution solution and 40 μL of blood is added) as the 100% hemolysis tube. Calculate the percentage of hemolysis for each tube. Report the result as the NaCl concentration that produces 50% hemolysis.
Table 1: Parpart NaCl Dilution Table and Hemolysis Rate
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6. Calculation of Results and Reference Range
Hemolysis Percentage (%) = (Absorbance of each tube / Absorbance of 100% hemolysis tube) × 100
Reference Range: 50% hemolysis concentration – 4.0 to 4.45 g/L
Notes
1. Each assay should include a normal control. A difference of 0.4 g/L in NaCl concentration between the normal control and the test sample is considered diagnostically significant.
2. Parpart NaCl Solution is prepared from high‑purity sodium chloride. Avoid contamination by acids or alkalis and store tightly sealed.
3. Blood samples should be added directly into the liquid; do not allow them to flow down the tube wall.
4. Do not use citrate or double oxalate as anticoagulants, as they may increase ionic strength and affect the osmotic pressure of the solution.
5. Use the reagents as soon as possible after opening to avoid affecting subsequent experimental results.
| R1507805 | Component | 100T | Storage |
| R1507805A | Parpart NaCl Solution | 30 mL | RT. |
| R1507805B | ddH₂O | 500 mL | RT. |
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