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. Hemoglobin C is an inherited mutation in the ß-globin gene. Persons of West African descent are much more likely to carry this mutation, which is the third most common variant worldwide.
Hemoglobin C is a normally-functioning oxygen carrier with a tendency to crystalize at normal oxygen tension. The peripheral smear of persons with C trait (a single mutated gene) is characterized by occasional target cells. Apart from a mild microcytosis, there are no other clinical complications. Homozygous hemoglobin C (2 mutated genes) is relatively common and presents with significant hemolysis with a reduced red blood cell (RBC) lifespan of 30-35 days. Dense polyhedral crystals can be seen both intracellularly and extracellularly on the peripheral smear. Most patients adapt well to the hemolysis, and anemia is often only borderline anemia; however, there is generally some splenomegaly present. Carriers of the mutation are usually only revealed through family studies or incidentally when testing for some other reason.
Significant anemia or pronounced microcytosis (<70 fL) with hemoglobin C should prompt further investigations for the coinheritance of a ß-thalassemia or of sickle hemoglobin, which is relatively common since the S mutation also occurs with high frequency in persons of West African descent (see chapter on Anemia Associated with Hemoglobin S-C).
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 RBC indices, a sickling test, plus 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 the sickling test is positive, a variant hemoglobin is suspected from HPLC, or there is clinical suspicion of a hemoglobinopathy, iso-electric focusing (IEF) or electrophoresis (EP) of hemoglobin dimers (or less commonly free globin chains) should be ordered.
Follow up tests on patients with known hemoglobin C requires only RBC indices, with quantitation of the known variant with HPLC or CEP.
Assessment of iron status is important in anemia and is usually accomplished through tests for ferritin and transferrin saturation (<20 ng/mL and <15%, respectively, in uncomplicated iron deficiency). (Table 1)
|Presumptive Diagnosis||Ratio Hemoglobin D/A||Hemoglobin A2 %||Other Hgb Present at >9 months of age||Sickling Test||Anemia||MCV/Morphology|
|C Trait||40/60||<3.7||F < 2%||negative||no||75 fL target cells|
|C/C Disease||100/0||<3.7||F < 3%||negative||mild to moderate hemolysis, which may be compensated||70 fL polychromasia, abundant target cells and spherocytes, anisocytosis HgbC crystals|
|C/A ß0 C/C ß0||100/0||>4.0 (unless the deletion is δß)||F > 5%||negative||Hgb 8-11 g/dL||55-70 fL polychromasia, abundant target cells and spherocytes absent crystals MCV/RBC < 14%|
|C/C ß+||100/0||>3.7||F < 3%||negative||Hgb 10-12 g/dL||55-70 fL MCV/RBC < 14%|
|C/A ß+||60-80/20-30||>3.7||F = 2-5%||negative||Hgb 11-13 g/dL||55-70 fL target cells MCV/RBC < 14%|
|Uncomplicated S/C||100/0||>4.5||S and C in equal amounts F > 5%||positive||Hgb 7-10 g/dL||normal|
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 arriving at 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 determine if iron deficiency is also present. In inflammatory disease, C-Reactive Protein is elevated.
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 percentages of hemoglobins A and C.
What Lab Results Are Absolutely Confirmatory?
The demonstration of substitution of lysine for glutamic acid at position 6 of the ß-globin chain is diagnostic for hemoglobin C (ß6 Glu → Lys). This is the same location as hemoglobin S, so, when SC is present, these mutations must be 1 on each globin chain. The expense of this test is rarely justified.
In practice, however, the demonstration of a peak on HPLC in the C-elution window, together with a band eluting with hemoglobin C on EP or IEF, is considered confirmatory for the presence of hemoglobin C. Presence of dense polyhedral crystals both intracellularly and extracellularly on the peripheral smear is also characteristic. Providing that the percentages of hemoglobin F and A2 are normal and the clinical severity is as expected, further testing is not usually warranted.
Hemoglobin C crystals are less commonly seen in C/Cß0 because of the reduced production of ß-chains.
In hemoglobin C trait, the percentage of hemoglobin C is always lower than that of hemoglobin A (typically 35-40%), but there is not a reduced rate of production of hemoglobin C ß-chains. The difference occurs because there is reduced affinity of the mutant ß-globin chains for α-globin chains. The precise reason for the hemolysis is unclear and may be a combination of the intra-erythroid crystal formation or cellular dehydration due to increase potassium eflux.
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 C to hemoglobin A will be observed as in adults.
Since hemoglobin S produces sickling only under reduced oxygen tension, a negative sickling test should be observed with hemoglobin C.
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 C may be indicated if these techniques are unable to arrive at 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). Because of the geographic distribution of α-thalassemia, this combination is unlikely. An elevated percentage of hemoglobin A2 is indicative of a ß-thalassemia. An elevated percentage of hemoglobin F is suggestive of a ß0 thalassemia (C ß-thalassemia is also seen in Mediterranean populations), hemoglobin S C disease, or hereditary persistence of fetal hemoglobin (see chapter on Anemia Associated with Hemoglobin S-C).
Treatment with 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?
Hemoglobins E, O-Arab, and C-Harlem all migrate with hemoglobin C on alkaline electrophoresis, but none of these coelutes with hemoglobin C on acid EP. On HPLC, there is, additionally, a small peak eluting slightly in front of the C peak, which is thought to represent glycated hemoglobin C.
The current generation of hemoglobin A1C (glycated hemoglobin) assays have eliminated previously observed unreliability in the presence of hemoglobin C trait, and results of diabetes tests can be interpreted with confidence in these patients.
The Sickling test is a screening test that detects any hemoglobin that polymerizes under reduced oxygen tension and cannot differentiate between Homozygous S disease, 1 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 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, which may need additional investigations, such as:
dietary iron deficiency or inadequate absorption (achlorhydria)
The following lab 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|
|sTfR / Log Ferritin||<1||>2||>2|
|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?