At a Glance
A family history of anemia in the absence of iron deficiency should prompt consideration of a hemoglobinopathy, and a number of these appear in the differential diagnosis. The hemoglobin O-Arab is an inherited mutation in the ß-globin gene. Persons of Balkan or North African black descent are more likely to carry this mutation. It is rarely found in Arabs.
Hemoglobin O-Arab is a stable, minimally increased oxygen affinity variant hemoglobin, and O-Arab trait (1 mutated gene) is completely silent. Homozygous hemoglobin O-Arab (2 mutated genes) is rare and is only occasionally reported to cause a very mild hemolytic anemia with some splenomegaly. Most patients adapt well to persistent borderline anemia, and the mutation is only detected incidentally during testing for some other reason. Carriers of the mutation are then revealed through family studies.
Significant hemolysis and/or anemia or microcytosis with hemoglobin O-Arab should prompt further investigations for the coinheritance of a thalassemia or sickle hemoglobin (see chapter on Anemia Associated with hemoglobin S-O Arab).
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, plus cation exchange high performance liquid chromatography (HPLC) or capillary electrophoresis (CEP).(Table 1)
If RBC indices are abnormal, it is appropriate to order morphology.
Always attempt to obtain a transfusion history.
If the sickling test is positive, a variant hemoglobin is suspected from HPLC, or there is clinical suspicion of a hemoglobinopathy, isoelectric focusing (IEF) or electrophoresis (EP) of hemoglobin dimers (or less commonly free globin chains) should be ordered.
Follow-up of patients with known hemoglobin O-Arab requires only RBC indices, with quantitation of the known variant with HPLC or CEP.
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).
|Presumptive Diagnosis||Ratio Hgb O-Arab/Hgb A||Hgb A2 %||Hgb F% at >9 months of age||Sickling Test||Hgb g/dL||MCV fL||Morphology|
|A/O-Arab||40/60||<3.7||1-3||negative||11-15||80-100||rare target cells|
|O-Arab/A ß+||70-80/15-20||>3.7||3-5||negative||9-11||65-72 MCV/RBC < 14||many target cells poikilocytosis|
|O-Arab/A ß0||100/0||>3.7||3-8||negative||6-8||50-70 MCV/RBC < 14||reticlocytosis many target cells marked splenomegaly|
|Uncomplicated S/O-Arab (see chapter on Anemia Associated with Hemoglobin S-O Arab)||100/0 S and O-Arab in equal amounts||<3.7||5-17||positive||8-11||80-95||reticulocytosis many target cells, poikilocytosis, anisocytosis normoblasts few sickle cells|
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?
Evaluation of the relative percentages of hemoglobin species detected is important to obtaining the correct diagnosis.
Iron deficiency can lower the percentage of hemoglobin A2, such that a ß-thalassemia could be overlooked. An MCV/RBC less than 14 is highly suggestive of ß-thalassemia. If using the value of hemoglobin A2 as a key indicator of ß-thalassemia, it is crucial to exclude the presence of A2′. This delta chain variant is clinically benign, but will be present at equal concentration to the A2, and must be added to it. It can be difficult to visualize on EP or IEF, since the percentage is small and it coelutes with hemoglobin S on HPLC.
Anemia of inflammation (anemia of chronic disease) has a normal/elevated ferritin, and further tests might be indicated to see if iron deficiency is also present. In inflammatory disease, C-Reactive Protein is elevated. It is important to determine the iron status of the patient who has O-Arab/thalassemia or S/O-Arab before supplementation or transfusion is given, as these can be iron loading. An iron overloaded patient typically has a transferrin saturation greater than 75%.
Transfused blood is always assumed to comprise 100% hemoglobin A, but this is not always the case as patients who are heterozygous for hemoglobin C or D mutations are not identified during donation, and this could alter the expected percentage of hemoglobin A and thus complicate the picture.
What Lab Results Are Absolutely Confirmatory?
The demonstration of substitution of lysine for glutamic acid at position 121 of the ß-globin chain is diagnostic for hemoglobin O-Arab (ß121 Glu → Lys). The expense of this test is rarely justified.
In practice, however, the demonstration of a peak on HPLC fractionally earlier than hemoglobin C, together with a band eluting in the region of hemoglobin A2 (where hemoglobins E and C-Harlem also elute) on IEF or alkaline EP, or between hemoglobin S and A on acid EP, is considered confirmatory for the presence of hemoglobin O-Arab. Hemoglobin C-Harlem gives a positive Sickling test and can easily be excluded. Additionally, hemoglobin O-Arab produces a characteristic, small peak in the S window on HPLC not seen with hemoglobin C. Providing that the percentages of hemoglobin F and A2 are normal, further testing is not usually warranted.
In hemoglobin O-Arab trait, the percentage of hemoglobin O-Arab is always lower than that of hemoglobin A (typically 40%), but there is not a reduced rate of production of hemoglobin O-Arab ß-chains. Interestingly, the point of the amino acid substitution in O-Arab is the same as in hemoglobin D-Los Angeles: (ß121 Glu → Lys) versus (ß121 Glu → Gln), respectively. This represents a critical point for tetramer assembly, and the resultant hemoglobin is slightly unstable, resulting in mild hemolysis in both variants (see Anemia Associated with Hemoglobin D-Los Angeles).
Many Newborn Screening programs include tests for common hemoglobinopathies. Once the percentage of hemoglobin F is subtracted from the total hemoglobin, the same ratio of hemoglobin O-Arab to hemoglobin A will be observed as in adults. This test will successfully identify hemoglobins F and A but may have difficulty identifying the O-Arab correctly.
What Confirmatory Tests Should I Request for My Clinical Dx? In addition, what follow-up tests might be useful?
Sequencing of the chromosome for the known specific point mutations for ß-globin O-Arab may be indicated if these techniques are unable to obtain a definitive diagnosis because of coelution with another hemoglobin.
Sequencing of the chromosome for common point mutations or deletions in the ß-globin gene is only rarely indicated in the event of the appearance of a previously unknown hemoglobin entity.
If the severity of the clinical presentation does not match the initial diagnosis, sequencing of the α- and/or ß-globin transcription regulator genes or sequencing of the gene in its entirety may be necessary to obtain a definitive diagnosis. The presence of hemoglobin H may indicate a 3-gene α-thalassemia (or a 2-gene deletion in a neonate). An elevated percentage of hemoglobin A2 is indicative of a ß-thalassemia. An elevated percentage of hemoglobin F is suggestive of a ß0 thalassemia, hemoglobin S/O-Arab disease (see Anemia Associated with Hemoglobin S-O Arab) or hereditary persistence of fetal hemoglobin.
Tests for hemolysis are only indicated in patients who are S/O-Arab or have a concurrent thalassemia.
Treatment with the hydroxyurea increases the percentage of hemoglobin F present. Hydroxyurea is given for polycythemias, sickle disease, and as a chemotherapeutic agent.
What Factors, If Any, Might Affect the Confirmatory Lab Results? In particular, does your patient take any medications – OTC drugs or Herbals – that might affect the lab results?
Hemoglobin O-Arab is sometimes confused with hemoglobin C, as it has very similar characteristics on some HPLC systems and on IEF. Hemoglobn C is also a variant ß-chain, which results from a substitution of lysine for glutamic acid at position 6, and is also clinically silent in the heterozygous state. The distinction between these two variants is important, especially if hemoglobin S is present, because the combination of S and O-Arab presents a much more severe clinical picture than hemoglobin S-C disease (see chapters on Anemia Associated with S-O Arab and Anemia Associated with Hemoglobin S-C).
Care should be taken when interpreting hemoglobin A1C (or glycated hemoglobin) in patients with hemoglobin O-Arab trait. Although most manufacturers provide good data for S and C variants, a number of HPLC methods are inaccurate with hemoglobin O-Arab trait; immunoassays seem more reliable.
The Sickling test is a screening test that detects any hemoglobin that polymerizes under reduced oxygen tension and cannot differentiate between Homozygous S disease or one of the sickle traits or the presence of a doubly substituted S mutation, such as hemoglobin C-Harlem. All results should be confirmed by additional testing, especially if they do not agree with the clinical picture.
The Sickling test may give a false negative if the hemoglobin S concentration is below 1 g/dL (typically <10-15% of the total hemoglobin), after transfusion, or in cases where the hemoglobin F is greater than 90% (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, such as multiple myeloma.
There are many causes of hemolysis other than hemoglobinopathies, some of which are:
RBC enzyme deficiencies, such as G6PD, Pyruvate Kinase, Glucose Phosphate Isomerase, or NADH reductase
mechanical destruction from artificial valves or burns
immunopathologic, such as transfusion reactions, Rhesus/ABO incompatibility, or warm and cold agglutinins
Tests indicative of hemolysis include decreased or absent haptoglobin, elevated LDH and unconjugated bilirubin, and elevated serum free hemoglobin.
There are many other common causes of anemia that may need additional investigations, such as:
dietary iron deficiency or inadequate absorption (achlorhydria)
The following laboratory tests help distinguish between anemia resultant from iron deficiency (IDA), inflammation (ACI), or concurrent iron deficiency with inflammation.(Table 2)
|Lab Test||ACI||IDA||IDA and ACI|
|Soluble Transferrin Receptor (sTfR)||normal||increase||normal/increase|
|Inflammatory Markers (CRP)||elevated||normal||elevated|
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- At a Glance
- What Tests Should I Request to Confirm My Clinical Dx? In addition, what follow-up tests might be useful?
- 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?
- What Confirmatory Tests Should I Request for My Clinical Dx? In addition, what follow-up tests might be useful?
- What Factors, If Any, Might Affect the Confirmatory Lab Results? In particular, does your patient take any medications - OTC drugs or Herbals - that might affect the lab results?