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 G-Philadelphia can arise from one of two different mutations in the α-globin gene. Although both produce the same protein, the mutations occur in different ethnic groups and produce different patterns of abnormal hemoglobins.

In the United States, G-Philadelphia is the most common α-chain variant and is seen in persons of African descent at a carrier rate of 1 in 5000. In this ethnic group, it is an inherited mutation on a previously mutated alpha gene and, therefore, has a thalassemic picture. Hemoglobin S and C ß-chain mutations are well represented in this population, which can lead to a bewildering array of hemoglobin species when coinherited. The Italian/Mediterranean mutation occurs on a normally functioning α-gene and is benign even when present in homozygous form.

Hemoglobin G-Philadelphia is a stable, normally-functioning oxygen carrier. G-Philadelphia trait (1 mutated gene) is completely silent. Homozygous G-Philadelphia (2 mutated genes) is rare and, depending on the origin of the mutation and the coinheritance of other alpha thalassemic mutations, produces a spectrum of effects from mild microcytosis to full blown H disease in very rare cases (see chapter on Alpha Thalassemia). Most patients without additional thalassemias adapt well to persistent borderline anemia, and the mutation is only detected incidentally during testing for some other reason. Carriers of both the G-Philadelphia mutations are then revealed through family studies.

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Significant hemolysis and/or anemia or microcytosis with hemoglobin G-Philadelphia trait should prompt further investigations for the coinheritance of a thalassemia or sickle hemoglobin (see chapters on Beta Thalassemia and Sick Cell Anemia).

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 tests of patients with known hemoglobin G-Philadelphia depends on the severity of the disease and may range from simple annual physicals to constant monitoring of RBC indices and iron status, with quantitation of the known variant with HPLC or CEP in transfusion dependent patients.

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).

Table 1
Presumptive Diagnosis Alpha Gene Arrangement Functioning α-genes Inheritance Hgb G, % Clinical Presentation
uncomplicated G trait, no thalassemia αG α/α α 4 Mediterranean 20-25 No defect
alpha thal 2 with trans G trait α -/αG – or – α/αG – 2 Mediterranean 35-45 Microcytic
alpha thal 2 trait , G trait α3.7G/α α 3 African American 25-35 Usually no defect
alpha thal 2, G trait α3.7G/- α 2 African plus other 40-45 MicrocyticHypochromic
alpha thal 2, homozygous G α3.7G/α3.7G 2 African American 95 MicrocyticHypochromic
α thal 2/α thal 1, G trait (also known as H Disease, G trait) α3.7G/ – – 1 rare no A Microcytic (MCV 55 fL) precipitation of beta-tetramers splenomegaly ineffective hematopoiesis bone marrow erythroid expansion
Alpha thal 2, G trait with ßS trait or ßC trait α3.7G/α α plus S or C 3 African American 25-35 SG or CG See chapter on Anemia Associated with Hemoglobin S-Beta Thalassemia

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?

The G-Philadelphia variant α-globin chain is seen with 2 different modes of inheritance. Two chromosomes each carry 2 genes for alpha chain production (α2 each produce 35% and α1 15% of the total alpla chains).

In Italians and those of Mediterranean descent, there is a simple, single-base substitution in codon 68 (AAC → AAA) on the α2 gene, resulting in a substitution of Lysine for Asparagine. This genotype has 4 normally functioning α-genes, 3 producing wild type, and 1 α2 producing G variant chains.

In contrast, African Americans show a quite different pattern. At some earlier time, there was a 3.7 kb deletion between the 3′ end portion of the α2 and the 5′ end of the α1 genes, resulting in a single α-gene on one chromosome (α2 thal, or -α3.7). At a later time, a second mutation occurred involving a single base substitution in codon 68 of this rearranged α2 gene (AAC → AAG), which again results in a substitution of Lysine for Asparagine. However, now there is also loss of one functioning α-gene, so this acts as an α-thalassemia. Given the frequency of this mutation in the African American population (28%), the inheritance of a second -α3.7 is significant.

Evaluation of the relative percentages of hemoglobin G species detected is important to arriving at the correct diagnosis. Since there is no preference in binding of the beta, gamma, or delta chains with the wild type or G variant alpha chains, the amount of each hemoglobin produced can be inferred directly from the percentage of G present. This greatly facilitates the diagnosis of the correct degree of α-gene compromise.

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 add the percentage of A2G, the variant α2Gδ2. This will be present in the same concentration ratio as the A is to the G. It can be difficult to visualize on EP or IEF, since the percentage in adults is small and it elutes 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.

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 complicate the picture.

What Lab Results Are Absolutely Confirmatory?

The demonstration of substitution of Lysine for asparagine at position 68 of the α-globin chain is diagnostic for hemoglobin G-Philadelphia (α68 Asn → Lys). The expense of this test is rarely justified.

The variety of hemoglobin species is always larger in an alpha chain variant, since alpha chains are constituents of hemoglobins A, A2, and F. In G-Philadelphia trait, these can be identified as:

α2 ß2 hemoglobin A

α2G ß2 hemoglobin G-Philadelphia

α2 δ2 hemoglobin A2

α2G δ2 hemoglobin A2G

α2 γ2 hemoglobin F

α2G γ2 hemoglobin FG

In practice, however, the demonstration of a peak on HPLC in the D-elution window, together with a band eluting with hemoglobin A on acid EP or with S on alkaline EP or on IEF, is considered confirmatory for the presence of hemoglobin G-Philadelphia. It can usually be differentiated from D by the presence of additional peaks on HPLC: FG just prior to A, and G2 in the S window. A2 will be decreased, since some of the delta chains are contained in G2. Providing that the percentages of hemoglobin F and A2 are as expected, further testing is not usually warranted.

In hemoglobin G-Philadelphia trait, the percentage of hemoglobin G is always lower than that of hemoglobin A (typically 25-40%), because only 1 of 4 genes are affected. Hemoglobin G-Philadelphia is stable, and hemolysis only occurs when there is a 3-alpha gene deletion and hemoglobin H is seen.

Many Newborn Screening programs include tests for common hemoglobinopathies, and, if the percentage of hemoglobin F is still very large, it may not be possible to firmly identify the small amounts of so many variant species. However, once the percentage of hemoglobin F is declined somewhat and is subtracted from the total hemoglobin, the same ratio of G to A will be observed as in adults. In neonates, FG will also be observed. On HPLC, this runs just prior to hemoglobin A.

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 α-globin G-Philadelphia may be indicated if these techniques are unable to arrive at a definitive diagnosis because of coelution with another hemoglobin.

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 arrive at 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 SG disease, or hereditary persistence of fetal hemoglobin.

Sequencing of the chromosome for common point mutations or deletions in the α-globin gene is only rarely indicated in the event of the appearance of a previously unknown hemoglobin entity.

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?

Interpreting hemoglobin A1C (or glycated hemoglobin) results in patients with hemoglobin G-Philadelphia is not usually problematic, since the targeted sites of glycation are on the beta-chains and these are not mutated, unless S or C is coinherited. Most manufacturers provide good data for S and C variants; 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 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)


chronic disease



GI bleeding

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

Table 2
Lab Test ACI IDA IDA and ACI
Transferrin decrease/normal increase decrease
Transferrin Saturation decrease decrease decrease
Ferritin normal/increase decrease decrease/normal
Soluble Transferrin Receptor (sTfR) normal increase normal/increase
sTfR/Log Ferritin <1 >2 >2
Inflammatory Markers (CRP) elevated normal elevated