Anemia Associated with Hemoglobin C

Table 1

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

infection

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)

pregnancy

chronic disease

malignancy

malnutrition

GI bleeding

The following lab tests help distinguish between anemia resultant from iron deficiency IDA), inflammation (ACI), or concurrent iron deficiency with inflammation. (Table 2)