Sickle Cell Screening Procedure

Principle:-

     The sickle screen kit provides a procedure based on hemoglobin solubility. Hemoglobin S is insoluble when combined with a buffer and a reducing agent. This occurs when the blood is mixed with the buffer and sodium hydrosulfite solution. Cells containing hemoglobin S are insoluble and show a turbid cloudy solution. Normal adult hemoglobin A is soluble and produces a transparent solution. The presence of hemoglobin S in either the heterozygous or the homozygous state produces a cloudy solution. Because this is a qualitative screening procedure, all positive results must be followed up with hemoglobin electrophoresis at  alkaline or acid pH or isoelectric focusing.

Reagents and Equipment:-

1. Sickle cell kit.

a. Phosphate buffer/sodium hydrosulfite solution. Prepare by pouring entire contents of sodium hydrosulfite vial into one phosphate buffer bottle. Cap and mix for 1 to 2 minutes. Once reconstituted, reagent is good for 5 days when stored at 2 to 8°C.

b. Unmixed reagents are good until expiration date on package when stored at 2 to 8°C.

2. 12-mm 75-mm test tubes.

3. Test tube caps or parafilm.

4. 50-μL pipette and tips.

5. Reading rack.

6. 5-mL pipette (to pipette the buffer).

Specimen Collection and Storage:-

1. Whole blood obtained in EDTA, heparin, or sodium citrate.

2. Specimens can be refrigerated at 2° to 8°C for 2 weeks before testing.

Quality Control:-

Commercially prepared negative and positive controls are run along with the patient’s blood. Control results must be correct to report patient results.

Procedure:-

1. Pipette 4 mL of the phosphate buffer/sodium hydrosulfite solution to each test tube (one for each test and each control).

2. Add 50 μL of well-mixed whole blood or control to each labeled tube.

3. Cover each tube with a cap or parafilm, and invert to mix three or four times.

4. Place each tube in the reading rack at room temperature, and let the tubes incubate for 10 to 20 minutes.

Interpretation of Results and Result Reporting:-

Positive: If hemoglobin S or any other sickling hemoglobin (hemoglobin C Harlem) is present, the solution will be turbid and the lines on the reading rack will be invisible.
Negative: If no sickling hemoglobin is present, the lines on the reading rack will be visible through the solution (figure below).

 Limitations:-

1. Severe anemias can cause false-negative results. The  sample volume should be doubled (100 μL) if the hemoglobin is less than 8 g/dL.

2. False-negative results can occur with infants younger than 6 months because hemoglobin F is insoluble in the test solution. Therefore, testing should not be done on infants.

3. Patients with multiple myeloma, cryoglobulinemia, and other dysglobulinemias may show false-positive results because the high protein level may affect the test.

4. Some rare hemoglobin variants such as hemoglobin C Harlem or C Georgetown may give false-positive results. These are sickling hemoglobins but do not contain hemoglobin S.

5. Patients who have been recently transfused may give false-positive or false-negative results.

6. Patients who have sickle trait give positive results. These results should be confirmed with hemoglobin electrophoresis.

7. Positive results and questionable results should be confirmed with hemoglobin electrophoresis.