Sickle Cell Procedure Principle

The sickle screen kit provides a procedure based on differential 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 hydrosulfate solution. Specimens 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 homozygous state will produce a cloudy solution. Because this is a qualitative screening procedure, all positives need to 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. Reagent, once reconstituted 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 X 75-mm test tubes

3. Test tube caps or parafilm

4. 50-pL pipette and tips

5. Reading rack

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 up to 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 pL of well-mixed whole blood or control to each labeled tube.

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

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

Interpretation of Results and Result Reporting

Positive: If hemoglobin S is present (or any other sickling hemoglobin [hemoglobin C Harlem]), the solution will be turbid and the lines on the reading rack are not visible.

Negative: If no sickling hemoglobin is present, the lines on the reading rack will be visible through the solution (Fig. 20.10).

Figure 20.10 Tube solubility screen for hemoglobin S. The procedure simply gives an indication of the presence of hemoglobin S and should be followed up with electrophoresis.

Figure 20.10 Tube solubility screen for hemoglobin S. The procedure simply gives an indication of the presence of hemoglobin S and should be followed up with electrophoresis.

Limitations

1. Severe anemias can cause false negatives. Therefore, if the hemoglobin is less than

8 g%, the sample volume should be doubled (100 pL).

2. False negatives can occur with infants under 6 months, because hemoglobin F is insoluble in the test solution. Therefore, testing on infants up to 6 months should not be done.

3. Patients with multiple myeloma, cryoglobulinemia, and other dysglobulinemias may give 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. Confirm these with hemoglobin elec-trophoresis.

7. Positive results and/or questionable results should be confirmed with hemoglobin elec-trophoresis.

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