Sickle Cell Anemia

Table 2

What Lab Results Are Absolutely Confirmatory?

In practice, the demonstration of a positive sickling test, a peak on HPLC in the S-window, and a band in the hemoglobin S position on EP or IEF is considered confirmatory for the presence of hemoglobin S. The demonstration of a substitution of valine for glutamic acid at position 6 of the β-globin chain is diagnostic for hemoglobin S (β6Glu→Val). This can be confirmed by genetic testing, although the expense of this test is rarely justified. The sickle mutation is at the same location as hemoglobin C, so when both hemoglobin S and C are present, these mutations must be one on each β-globin gene.

In sickle cell anemia, hemoglobin S comprises 90-95% of the total hemoglobin, with the remaining hemoglobin being hemoglobin F and A2. Deviation from this pattern is suggestive of a different hemoglobinopathy, a concurrent α- or β-thalassemia, and/or iron deficiency and requires further investigation.

Many newborn screening programs include tests for common hemoglobinopathies and successfully identify hemoglobin S. The expected pattern will be F-S. The presence of any hemoglobin A suggests an alternative hemoglobinopathy.

What Tests Should I Request to Confirm My Clinical Dx? In addition, what follow-up tests might be useful?

If the severity of the clinical presentation does not match the initial diagnosis, sequencing of the α- and/or β-globin genes, including up-stream regulatory sequences, may be necessary to arrive at a definitive diagnosis. The presence of hemoglobin H may indicate a 3-gene α-thalassemia (or a 2-gene α-thalassemia in a neonate). An elevated percentage of hemoglobin A2 is indicative of a β-thalassemia in a nondiabetic patient.

An elevated percentage of hemoglobin F is suggestive of an α-thalassemia, β-thalassemia, hemoglobin S/C disease, or hereditary persistence of fetal hemoglobin. Treatment of hydroxyurea increases the percentage of hemoglobin F and has an ameliorating effect on sickling. It is also used for polycythemias and as a chemotherapeutic agent. Finally, infants with sickle cell anemia may maintain elevated concentrations of hemoglobin F for about 2 years before attaining adult concentrations (compared to 6 months in unaffected infants). Monitoring the percentage of hemoglobin F present may be useful for hydroxyurea therapy.

Patients with sickle cell anemia may become folate-deficient because of the increased erythrocyte turnover that results from hemolysis. Serum or RBC folate should be monitored and supplemented as necessary, or folate could be used prophylactically. Folate deficiency exacerbates the anemia.

High levels of hemolysis can lead to icterus and possible cholelithiasis; monitoring of bilirubin and alkaline phosphatase is indicated to minimize risk hepatic damage.

Are There Any Factors That Might Affect the Lab Results? In particular, does your patient take any medications – OTC drugs or Herbals – that might affect the lab results?

Hemoglobins D-Los Angeles and G-Philadelphia migrate with hemoglobin S on some EP methods, but not HPLC; neither gives a positive sickling test.

The current generation of hemoglobin A1C (glycated hemoglobin) assay has eliminated previously observed unreliability in the presence of hemoglobin S-trait, so A1C can be interpreted with confidence in these patients. However, most HPLC A1C assays, with the exception of boronate affinity chromotography, will not give reliable results in patients with sickle cell anemia; modern immunoassays or enzymatic assays for glycated hemoglobin perform well.

The sickling test is a screening tool that detects any hemoglobin that polymerizes under reduced oxygen tension. Therefore, this assay cannot differentiate between homozygous S and one of the sickle traits (e.g., hemoglobin SC). All results should be confirmed by additional testing, especially if they do not agree with the clinical picture. Where patients’ percentages of hemoglobins S, A, and F differ from those expected in uncomplicated presentations, considerations of alternative hemoglobin variants are important. Other hemoglobins that also give positive sickling tests may need to be considered. With the exception of C-Harlem, most are rare or isolated reports. With the exception of Porto-Alegre, all contain the S mutation (ß6 Glu → Val) in addition to the following mutations:

Hgb C-Harlem (ß73 Asp → Asn) (C-Georgetown)

Hgb C-Ziquinchor (ß58 Pro → Arg)

Hgb S-Oman (ß121 Glu → Lys)

Hgb S-Providence (ß82 Asn → Asp)

Hgb S-Travis (ß121 Ala → Val)

Hgb Jamaica Plain (ß68 Leu → Phe)

Hgb Antilles (ß23 Val → Ile)

Hgb Porto-Alegre contains only the (ß9 Ser → Cys) mutation

The sickling test may give a false negative if the hemoglobin S concentration is less than 1 g/dl (typically <10-15% of the total hemoglobin). This may occur following transfusions or in cases of high hemoglobin F (e.g., neonates and hereditary persistence of fetal hemoglobin).

The sickling test may give a false positive if there are nucleated RBCs in the peripheral blood or the patient has a marked hypergammaglobulinemia (e.g., multiple myeloma).

There are many causes of RBC hemolysis other than hemoglobinopathies, some of which include:

• RBC enzyme deficiencies (e.g., G6PD, pyruvate kinase, glucose phosphate isomerase, NADH reductase)
• mechanical destruction from artificial valves, burns
• infection
• immunopathology (e.g., transfusion reactions, Rhesus/ABO incompatibility, warm and cold agglutinins)

Tests indicative of hemolysis include decreased or absent haptoglobin, elevated LDH and unconjugated bilirubin, and elevated serum-free hemoglobin.

Other common causes of anemia to be considered include:

dietary iron deficiency or inadequate absorption (achlorhydria)

pregnancy

chronic disease

malignancy

malnutrition

GI bleeding