At a Glance
Sickle cell anemia, or homozygous hemoglobin S, may be expected in any person with anemia (hemoglobin < 0 g/dL), abnormal peripheral blood findings, a family history of sickle cell trait or sickle cell anemia, and/or a positive newborn screen for sickle cell anemia. The sickle mutation is most commonly found in persons of African, Middle Eastern, or Mediterranean descent. Approximately 8% of African Americans are carriers, and 1 in 375 has sickle disease. The term “sickle disease” is rather broad and descriptive of the morphology and can occur with double heterozygous states, including hemoglobin S trait, such as Hemoglobin C, D and O-Arab. These have been described in their respective chapters.
Hemoglobin S arises from a mutation in the β-globin gene, which decreases hemoglobin solubility in the de-oxygenated state. De-oxygenated mutant hemoglobin forms polymers that contour the red cell into the characteristic sickled shape. These stiff and dysmorphic cells can block microvasculature, causing vaso-occlusive events that contribute significantly to morbidity.
In addition to anemia, the peripheral blood smear reveals characteristic sickled and target cells, polychromasia, and signs of splenic damage, such as Howell-Jolly and Pappenheimer bodies.
Clinical findings can include hemolysis, anemia, vaso-occlusive pain crises, autosplenectomy, cerebrovascular accidents, skin ulcers, acute chest syndrome, and aseptic necrosis of the hip.
What Tests Should I Request to Confirm My Clinical Dx? In addition, what follow-up tests might be useful?
The standard hemoglobin evaluation for diagnostic purposes consists of red blood cell (RBC) indices, a sickling test, and either cation-exchange high-performance liquid chromatography (HPLC) or capillary electrophoresis (CEP).
If RBC indices are abnormal, it is appropriate to order morphology.
Always attempt to obtain a transfusion history.
If either the sickling test is positive or a variant hemoglobin is suspected, iso-electric focusing (IEF) or electrophoresis (EP) of hemoglobin dimers should be ordered.
Individuals with sickle cell anemia demonstrate normocytic anemia with abnormal RBC morphology (e.g., sickle and target cells, polychromasia), a positive sickling test, and 90-95% hemoglobin S by HPLC or CEP. The remainder of the hemoglobin will be comprised of hemoglobin F and A2. No hemoglobin A should be present in this condition.
Assessment of iron status is important in anemia, which is usually accomplished through tests for ferritin and transferrin saturation (<20 ng/mL and <15%, respectively, in uncomplicated iron deficiency). Sickle anemia is an iron-loading condition, particularly if transfusions are required. Ferritin should be monitored, and the value should be kept below 1,000 ng/mL by the use of chelators like Exjade (Table 1).
|Sickling test||Hemoglobin HPLC or CE|
|Positive||90-95% hemoglobin S|
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?
Iron deficiency can lower the percentage of hemoglobin A2, which may mask a concurrent β-thalassemia. An MCV/RBC less than 14 is highly suggestive of β-thalassemia.
If using the value of hemoglobin A2 has a key indicator of β-thalassemia, it is crucial to exclude the presence of hemoglobin A2′. This delta chain variant is clinically benign but will be present at equal concentration to hemoglobin A2; to obtain an accurate value of delta chain concentrations, hemoglobins A2 and A2′ must be added together. It can be difficult to visualize hemoglobin A2′ on EP or IEF since the percentage is small and it coelutes with hemoglobin S on HPLC.
Glycated hemoglobin S elutes with hemoglobin A2 on HPLC and may falsely elevate the value of hemoglobin A2, leading to erroneous suspicion of β-thalassemia.
Finally, β-thalassemias may mask the presence of a mutant hemoglobin if the thalassemia completely suppresses expression of the mutant gene. It is important to ascertain the correct disease state, as the disease state can have significant implications for future generations. If the elevated hemoglobin A2 is not recognized as β-thalassemia, offspring with a partner with sickle cell trait could unexpectedly have severe hemoglobin S/β0 disease, rather than benign sickle cell trait.
Transfusion is always assumed with 95% hemoglobin A, although occasionally hemoglobin C- or D-trait blood is transfused, which results in unexpected hemoglobin variants. Many patients with sickle cell anemia are transfusion-dependent, and it is important not to assign the presence of some hemoglobin A to the S/β+ phenotype if the hemoglobin A came from transfused blood. Likewise, following a bone marrow transplant, sickle cell patients have varying amounts of hemoglobin A and S, depending on the hematological state of the donor and engraftment of the tissue.
Anemia of inflammation (anemia of chronic disease) has a normal to elevated ferritin, and further tests are required to determine whether iron deficiency is also present. Iron overload is indicated when the transferrin saturation is greater than 75%. If iron overload is already established, monitoring liver function may be indicated ,and the physician should be attuned to monitoring for other organ damage (Table 2).
|Laboratory Test||ACI||IDA||ACI and IDA|
|Soluble Transferrin Receptor (sTfR)||normal||increased||normal/increased|
|Inflammatory Markers (e.g., CRP)||elevated||normal||elevated|
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.
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.
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
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)
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- At a Glance
- 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?
- What Lab Results Are Absolutely Confirmatory?