Hematology test, Osmotic fragility erythrocytes

When erythrocytes are in solution hipotonis, osmolalitasnya fluid levels lower than normal plasma or serum (less than 280 mOsm / kg)

Erythrocyte osmotic fragility test (also called erythrocyte osmotic resistance) is done to measure the ability to withstand the occurrence of erythrocyte hemolysis (destruction of red blood cells) in asolution that hipotonis. The trick is as follows: erythrocytes were dissolved in saline solution with various concentrations. If hemolysis occurs at a slightly saline solution hipotonis, this state is called an increase in erythrocyte fragility (= reduction in resistance / durability erythrocytes), and if hemolysis occurs at a very hipotonis salinesolution, this situation indicates decreased osmotic fragility (= increase in erythrocyte resistance).

Hemoglobin out of the cells in each tube containing a solution of NaCl of different levels. Hb then determined fotokolorimetrik. The results are reported in percentages (%) hemolysis. Collection of hemolysis results are plotted in a curve as compared with normal erythrocytes data. In the state of increased fragility, erythrocytes are usually spherical, and the curves seem to shift to the right. While the decrease in fragility, thin and flat-shaped erythrocytes, the curve appears shifted to the left.

Clinical Problems

DECREASE fragility: Thalassemia major and minor (Mediterranean anemia or Cooley's anemia), anemia (iron deficiency, folic acid deficiency, vitamin B6 deficiency, sickle cell) hemoglobin C disease, polycythemia vera, post splenectomy, acute liver necrosis and sub-acute, jaundice obstructive.

IMPROVEMENT fragility: hereditary spherocytosis, transfusions (ABO and Rhesus incompatibility), autoimmune hemolytic anemia (AIHA), hemoglobin C disease, drug toxicity or chemicals, chronic lymphocytic leukemia, burns (thermal).

Procedure

This test is usually performed on fresh blood samples of less than 3 hours and / atu 24-hour blood samples were incubated at 37 ° C. Blood samples used in the form of heparin blood or blood "defibrinated". There are no restrictions on food or beverage intake.

In this test made solution with different concentrations of NaCl. Assessment of results with fotokolorimetri method (using photometer or spectrophotometer).

Prior to testing, provide a first buffer stock solution of NaCl 10% made from 9 grams NaCl, 1.365 g Na2HPO4, and 0.215 grams NaH2PO4.H2O. The material is then diluted with distilled water to 100 ml. Before being used for inspection, make asolution of 1.0% NaCl principal by dissolving 5.0 ml of saline buffer stock of 10% with distilled water to 50.0 ml. Next do the testing as follows :

1. Provide 12 pieces and then make a dilution tube multilevel solution of NaCl concentration: 0.85%, 0.75%, 0.65%, 0.60%, 0.55%, 0.50%, 0.45%, 0, 40%, 0.35%, 0.30%, 0.20% and 0.10%, respectively of 5.0 ml. NaCl solutions were made from the basicsolution of NaCl 1.0%.
2. Add into the canisters each 50 mL blood sample. Mix (homogenization) by way of tossing and turning the tube several times.
3. Inkubasikan for 30 minutes at room temperature.
4. Mix (homogenization) again and then worry about (centrifuges) of each tube for 5 minutes at 3000 rpm.
5. Measure the absorbance (OD) of the supernatant at λ 540 nm with the blank tube to supernatant-1 (0.85% NaCl).
6. Calculate the% hemolysis by dividing the absorbance (OD) of samples with the absorbance (OD) tubing to 12 multiplied by 100%.
7. Create a curve with the concentration of NaCl as the axis (x) and% hemolysis as the ordinate (y). Compare with the curve of normal blood controls.

Normal Value

The beginning of hemolysis on the concentration of NaCl 0.40% - 0.45%

Complete hemolysis at a concentration of 0.30% NaCl - 0.35%

The percentage of hemolysis in normal circumstances are :

97-100% hemolysis in 0.30% NaCl

50-90% hemolysis in 0.40% NaCl

5-45% hemolysis in 0.45% NaCl

0% hemolysis in 0.55% NaCl

Factors Affecting Laboratory Findings

• plasma pH, temperature, glucose concentration, and oxygen saturation in the blood
• Long-lived erythrocytes tend to have high osmotic fragility
• Blood samples taken over 3 hours can show increased osmotic fragility.

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