Sickling Test & Solubility test

• Sickling screening tests are part of the diagnostic work up for patients suspected of having a sickle cell syndrome and should be carried out if there is an abnormal electrophoretic haemoglobin fraction in the position of hemoglobin S.
• Sickle cell anemia (sickle cell disease) is a blood disorder caused by an inherited abnormal hemoglobin.
• Hemoglobin S results from the replacement of glutamic acid (negatively charged) by valine (hydrophobic amino acid) at position 6 of the beta globin chain of the hemoglobin molecule.
• The abnormal hemoglobin causes RBCs to assume an abnormal, rigid, sickle shape, making cells fragile and prone to rupture.
• Sickle cell trait individuals (sickle cell carriers) have one normal beta globin gene and one sickle cell gene with roughly equal proportions of usual adult hemoglobin and sickle hemoglobin.
• Sickle cell trait carriers do not experience any health problems as a result of having the trait, unlike individuals who inherit two sickle cell genes and have sickle cell disease.
• Severity of sickle-cell disease depends on:
  • Extent of oxygen loss where prolonged oxygen deprivation contributes to the severe pain experienced as a sickle-cell crisis.
  • The acidity of the environment, where organs that are most seriously affected are those with an acidic environment like the spleen and bone marrow (BM).
  • The concentration of Hb S within the cell, where the lower the better.

Sickling Test

• A drop of blood is mixed with one drop of sodium metabisulfite reagent on a slide, RBCs containing hemoglobin S will become sickle-shaped (half-moon-shaped).
• The reagent removes oxygen from the cells, allowing sickling to take place through complex polymerization reactions.
• Use EDTA anticoagulated whole blood.
• Use 2% fresh sodium metabisulfite (Na2S2O5) solution.
• Use a microscope, microscope slides, cover slips, filter-paper, pasteur pipette (or dropping pipette), two small wooden sticks and containers like Petri dishes to prevent drying.
• Procedure:
  • Grab a capillary blood drop (4mm diameter) and place in the centre of the slide.
  • Add an equal-sized drop of sodium metabisulfite solution.
  • Mix with the corner of a slide.
  • Cover with a cover slip, making sure that no air bubbles form.
  • Seal the edge with wax/vaseline mixture or with nail varnish.
  • Place the slide in a Petri dish that has wet filter-paper in the bottom and support the slide on two sticks.
  • Wait 30 minutes before examining the slide with the 40× objective lens on a microscope.
• Positive results show erythrocytes that become sickle-shaped or banana-shaped, often with spikes.
• Examine several parts of the preparation, as sickling can occur more quickly in one part than in another, mistaking normal erythrocytes lying on their side for sickle cells.
• Negative results show erythrocytes that remain round.
• If the test is negative, re-examine the slide after a further 30 min, then after 2 hrs and after 24 hrs.
• False-negative results may occur if; reagents have expired, concentrations of hemoglobin S are low (as in newborn cells), or if the cover slip is not sealed properly.
• This test cannot distinguish the sickle cell trait from sickle cell disease.

Solubility test

• 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 and it forms tactoids (water crystals) which produce a turbid solution.
• Note that I forms tactoids (water crystals)
• Use EDTA anticoagulated whole blood.
• Use phosphate buffer preparation with 215 g K2HPO4, 169 g KH2PO4, 5g sodium dithionite and 1g saponin, made up to 1 liter with distilled water and keep refrigerated for 7 days.
• Use three test tubes (12mm×75mm), pipettes (1000μL & 10μL) and lined paper.
• Procedure:
  • Pipette 2 ml of reagent into three test tubes. Allow reagent to warm to room temperature.
  • Add 10 µL of packed cells from the test blood sample to one tube.
  • Add 10 μL of packed cells from sickle cell positive blood to the second tube as a positive control.
  • Add 10µL of packed cells from normal blood to the third tube as a negative control.
  • Mix well and leave to stand for 5 minutes.
  • 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.
  • If the test appears to be positive, centrifuge at 1200g for 5 minutes.
• Positive tests prevent any lines being seen through the solution
• Negative tests allow lines that can be seen through the test solution
• After centrifugation, positive tests will show a dark red band at the top with pink or colorless solution below.
• False positive test results are attributed severe leukocytosis, hyperproteinemia and hyperlipidemia.
• False negative test results are attributed deteriorated reagent, infants less than 6 months of age, hemoglobin S less than 20% like post transfusion and patients with low hemoglobin.
• All sickle screening tests should be confirmed by electrophoresis.