Introduction

Haemoglobinopathies constitute a large proportion of hemolytic anemias constituting around 74% of hereditary hemolytic anemias (1). These cases are usually symptomatic, however in a significant proportion of patients they are detected incidentally. Structural hemoglobin variants mostly occur due to point mutations in the alpha or beta globin genes. About 200 alpha chain variants have been identified (2). Most of the variants have characteristic findings on High performance liquid chromatography (HPLC). A few may present as unknown peaks that may lead to diagnostic difficulty.

Case Report

A 53 year old male presented to the Medicine out-patient department with complaints of easy fatiguability, yellowish discoloration of sclera and high-colored urine. There was no other significant complaint or past history. On examination, there was no lymphadenopathy or hepatosplenomegaly.

A complete blood count was sent which revealed severe anemia with hemoglobin level of 5.6 gm/dL, mean corpuscular volume (MCV) 111.2 fL, mean corpuscular hemoglobin (MCH) 28.6 pg, mean corpuscular hemoglobin concentration (MCHC) 25.7 g/dL, and markedly raised reticulocyte count 26.8%. The total leucocyte count and platelets were in normal range. Peripheral smear revealed red blood cells showing marked anisopoikilocytosis with presence of microcytic hypochromic cells, few macrocytes, target cells, few elliptocytes and occasional spherocytes. Many polychromatophils and few nucleated red blood cells were also seen. (Figure 1)

On immune-hematological workup, Direct Coomb's Test was 2+ positive and Indirect Coomb's Test was negative. (Figure 2)

Figure 1: Peripheral smear showing microcytic hypochromic blood picture along with presence of few target cells, elliptocytes, spherocytes and polychromatophils. One nucleated red blood cell is also seen. [1000x, Wright -Giemsa stain] Inset shows marked reticulocytosis. [1000x, New Methylene Blue preparation]

Figure 2: Gel card showing negative antibody screening (I, II and III) (Indirect Coomb’s test negative) and 2+ positive direct Coomb’s test.

Figure 3: High performance liquid chromatography chromatogram showing the unknown peak at retention time 2.72 minutes, 12.3%

Due to high suspicion of hemoglobinopathy, High performance liquid chromatography (HPLC) was performed which revealed HbA₀ 76.9%, HbF 2.5% and HbA2 1.6%. An unusual peak was identified which appeared as a hump in the peak adjoining HbA₀ a with retention time of 2.72 minutes comprising 12.3% of the total Hemoglobin (Figure 3).

The different hemoglobin variants which elute in the A₀ window (Retention time = 1.90-3.10 minute) include 4 hemoglobin variants (2 alpha and 2 beta chain variants) Hb Ty Gard, Hb Köln, Hb Twin Peaks along with Hb Fontainebleau (3,4). Differentiation of these variants depends on the characteristic chromatogram findings [Table 1]. The peak seen in the index case was characteristic of Hb Fontainebleau. However, molecular analysis is needed for definitive diagnosis which could not be performed as the patient was lost to follow up.

Differential diagnosis of Hemoglobin variants eluting in the A₀ window (Retention time = 1.90-3.10 minute) (3) using Bio-Rad Variant II HPLC system (Beta thalassemia short program).

Discussion

Hb Fontainebleau is an alpha chain variant characterized by an Alanine → Proline substitution at codon 21 with a GCT>CCT change at the molecular level, this proline residue is located at the beginning of the alpha helix. Usually, replacement of any residue by Proline in the alpha helix leads to instability of the molecule. However, in Hb Fontainebleau the structural modification involves an external residue at the beginning of the chain. This is well accommodated without a change in the hemoglobin stability or the oxygen binding capacity.

The first case of Hb Fontainebleau was reported in an adolescent girl of Italian origin living in France. The variant was detected by Isoelectric focusing. (IEF). The patient had hemolytic syndrome since the age of 5 years. The severe phenotype was explained by a co-existing red cell membrane defect, Spherocytosis (25-28%) (5). The second case was described in an adult male of Iraqi origin in New Zealand who presented with microcytosis (27.5%) (6), the third case was from Cyprus and was detected during Thalassemia screening (7). It was first reported in India in a mother (RT= 2.90 min, 1.5%) and her newborn (RT= 2.90 min, 8.5%) in Madhya Pradesh both of whom had co-existing HbS (8). The variant was later also reported in Punjab in an adult female who came for routine ANC thalassemia screening with normal hematological indices but presented with bad obstetric history (RT = 2.89 min, 14.9%). Another case was detected in an adult male who presented with weakness and easy fatiguability. There was no hepatosplenomegaly or lymphadenopathy. HPLC revealed the peak at RT of 2.92 min, 13.8% which appeared as a hump in the preceding HbA peak. Molecular studies revealed G>C substitution at codon 21 of alpha 2 globin gene which leads to substitution of alanine with proline leading to a diagnosis of Hb Fontainebleau co-existing with Iron deficiency anemia (9).

The range of retention times found for Hb Fontainebleau in past literature varies from 2.89 – 2.92 minutes and the percentage of Hb Fontainebleau varies from 1.5% - 28%. In the present case RT = 2.72 minutes and the percentage of Hb Fontainebleau was = 12.3%. The current case was within range.

Clinically, Hb Fontainebleau on its own does not cause any symptoms usually. It is often detected as an incidental finding on HPLC screening. So, a patient with Hb Fontainebleau presenting with anemia should be worked up for a co-existing underlying cause. The current case with severe anemia on workup revealed DCT positivity and spherocytes on peripheral smear, suggesting that a component of Immune hemolytic anemia was present. Inability to perform family workup for similar hemoglobinopathy and lack of molecular analysis was a major shortcoming in this case. However, with the knowledge of the characteristic peaks, hemoglobin percentages and retention times on HPLC, most variant hemoglobins can be identified.

Thus, HPLC serves as a very useful and powerful diagnostic tool.

References

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