Screening Tests for Hemoglobin S PDF

**HAEMATOLOGY II** **HML2143** **[PRACTICAL 4]** **Screening Tests for Hemoglobin S** **Introduction** - Carrying out the sickling screening tests is part of the diagnostic work up in patients suspected of having a sickle cell syndrome. These tests should be also carried out if there...

**HAEMATOLOGY II** **HML2143** **[PRACTICAL 4]** **Screening Tests for Hemoglobin S** **Introduction** - Carrying out the sickling screening tests is part of the diagnostic work up in patients suspected of having a sickle cell syndrome. These tests should be also carried out if there is an abnormal electrophoretic haemoglobin fraction in the position of hemoglobin S. - Sickle cell anemia (sickle cell disease) is a disorder of the blood caused by an inherited abnormal hemoglobin. Hemoglobin S results from the replacement of an amino acid glutamic acid (negatively charged) by valine (hydrophobic amino acid) at position 6 of the beta globin chain of hemoglobin molecule. The abnormal hemoglobin causes RBC\'s to assume an abnormal, rigid, sickle shape cells, which are fragile and prone to rupture. - Individuals who have sickle cell trait (called sickle cell carriers) have one normal beta globin gene and one sickle cell gene. These individuals make both the usual adult hemoglobin and sickle hemoglobin in roughly equal proportions, so they do not experience any health problems as a result of having the trait. But, individuals who inherit two sickle cell genes have sickle cell disease. - The severity of sickle-cell disease generally depends on a number of factors: - The extent of oxygen loss: prolonged oxygen deprivation contributes to the severe pain experienced as a sickle-cell crisis. - The acidity of the environment: the organs most seriously affected are those with an acidic environment (such as the spleen and BM). - The concentration of Hb S within the cell: the lower the better. **Screening Tests for Hemoglobin S** A. **Sickling Test** **Principle** When a drop of blood is mixed with one drop of sodium metabisulfite reagent on a slide, RBCs containing hemoglobin S will become sickle-shaped or half-moon-shaped. The reagent removes oxygen from the cells, allowing sickling to take place through complex polymerization reactions. **Specimen** EDTA-anticoagulated whole blood **Reagents** 2% fresh sodium metabisulfite (Na~2~S~2~O~5~) solution. **Equipments** - Microscope - Microscope slides - Cover slips - Filter-paper - Pasteur pipette (or dropping pipette) - Two small wooden sticks - Containers to prevent drying of the preparation, such as Petri dishes **Procedure** 1. Place a small drop of capillary blood (about 4mm diameter) in the centre of a slide as shown below: 2. Add an equal-sized drop of sodium metabisulfite solution. 3. Mix carefully with the corner of a slide. 4. Cover with a cover slip, making sure that no air bubbles form. 5. Seal the edge with wax/vaseline mixture or with nail varnish. 6. Place the slide in a Petri dish that has wet filter-paper in the bottom. Support the slide on two sticks. 7. Wait 30 minutes before examining the slide. 8. Examine the slide under the microscope using the **[40×]** objective lens. **Results Interpretation** a. Positive result (figure 1): - The erythrocytes become sickle-shaped or banana-shaped, often with spikes. - It is important to examine several parts of the preparation, as sickling can occur more quickly in one part than in another. Do not mistake normal erythrocytes lying on their side for sickle cells.  **Figure 1:** Positive sickling test (a: Sickle-shaped erythrocytes, b: sickle shaped erythrocytes with spikes). ***Note:** A positive test **[does not]** distinguish the sickle cell trait from sickle cell disease* b. Negative result (figure 2): - The erythrocytes remain round. - If the test is negative, re-examine the slide after a further 30 min, then after 2 hrs and after 24 hrs. **Figure 2:** Negative sickling test. **Sources of Errors** False-negative results may occur if: - Expired reagents are used - Concentrations of hemoglobin S are low (as in newborn cells) - If the cover slip is not sealed properly B. **Solubility test** **Principle** - Erythrocytes are lysed by saponin and the released hemoglobin is reduced by dithionite in a phosphate buffer. - Hemoglobin S is quite insoluble when in the reduced state in high phosphate buffer solution. It forms tactoids (water crystals) which produce a turbid solution. **Specimen** EDTA-anticoagulated whole blood **Reagents** - Phosphate buffer preparation: - 215 g K2HPO4 - 169 g KH2PO4 - 5g sodium dithionite - 1g saponin - Make up to 1 liter with distilled water - Keep refrigerated for 7 days **Equipments** - Three test tubes (12mm×75mm) - Pipettes (1000μL & 10μL) - Lined paper - Centrifuge **Procedure** 1. Pipette 2 ml of reagent into three test tubes. Allow reagent to warm to room temperature. 2. Add 10 μL of packed cells from test blood sample to one tube. 3. Add 10 μL of packed cells from sickle cell positive blood to the second tube (positive control). 4. Add 10μL of packed cells from normal blood to the third tube (negative control). 5. **Mix well and leave to stand for 5 minutes.** 6. **Hold the tube 2.5 cm in front of white card with narrow black lines and read for turbidity, in comparison with the positive and negative control samples.** 7. **If test appears to be positive, centrifuge at 1200*g* for 5 minutes.** **Results (Figure 3)** - **Positive tests: lines on card cannot be seen through the test solution.** - **Negative test: lines on** card can be seen through the test solution. - After centrifugation, **positive tests will show**: Dark red band at the top with pink or colorless solution below. **Sources of Errors** a. False positive results: - Severe leukocytosis - Hyperproteinemia - Hyperlipidemia b. False negative results: - Deteriorated reagent. - In infants less than 6 months of age - Hemoglobin S less than 20% (*e.g*., post transfusion) - In patients with low hemoglobin ***Note:** All sickle screening tests should be confirmed by electrophoresis.*  **Figure 3:** Solubility test results ( A: Negative Solubility test: lines on card can be seen through the test solution, B; Positive Solubility tests: lines on card cannot be seen through the test solution).