The results of the investigations conducted are summarized in Table 1.

The laboratory investigations of this patient showed that the patient had mild microcytic hypochromic anaemia. However, since the iron studies were found to be normal, the physician decided to order an Hb HPLC study. The biochemist discussed the Hb HPLC findings (Figure 1)with the treating physician and screening of both parents was considered for confirmation of the findings. The patient’s father turned out to be a carrier of thalassaemia and the mother of Hb D-Punjab.Assessment of liver function showed mild unconjugated hyperbilirubinaemia withnormal serum proteins and liver enzymes. The workup for haemolysis was negative. Thus, a genetic analysis of the UGT1A1 polymorphism was conducted, revealing the patient's UGT1A1*28/*28 genotype, which is linked to a significant decrease in UGT enzyme activity.

Questions
1. What are the abnormal findings in Hb HPLC results in this patient?
2. What is the cause of hyperbilirubinemia in this patient?
3. What guidance ought to be provided to this patient?

Discussion

Question 1
Hb HPLC study revealed the presence of HbD, high HbF and normal HbA2 in this patient. HPLC findings are suggestive of compound heterozygous HbD-Punjab and beta thalassemia trait with a false normal HbA2. The screening of parents for

Question 2
The absence of evident hemolysis and underlying hepatic pathology indicates the possible presence of Gilbert syndrome. Genetic assessment of the UGT1A1 polymorphism confirms thatthe patient has the UGT1A1*28/*28 genotype that is associated with severely reduced UGT enzyme activity. This genetic profile strongly suggests that the primary factor contributing to unconjugated hyperbilirubinemia is most likely the reduced conjugation of bilirubin resulting from diminished activity of the UGT enzyme in this patient. Notably, a homozygous HbD state typically only results in sub-clinical jaundice, making Gilbert syndrome a major contributor to the observed hyperbilirubinemia.

Question 3
The patient needs to be counseled and reassured regarding the benign course of the disease. She should be sensitized about various precipitating factors like fever, physical exertion, prolonged fasting, etc. that may increase her bilirubin levels. The patient should receive guidance to refrain from undergoing unnecessary laboratory investigations to avoid thepotential discomfort or inconvenience associated with repeated blood draws.

1. Torres L de S, Okumura JV, Silva DGH da, Bonini-Domingos CR. Hemoglobin D-Punjab: Origin, distribution and laboratory diagnosis. Rev Bras HematolHemoter. 2015;37(2):120–6.

2. Basmanj MT, Karimipoor M, Amirian A, Jafarinejad M, Katouzian L, Valaei A, et al. Co-inheritance of hemoglobin D and β-thalassemia traits in three Iranian families: Clinical relevance. Arch Iran Med. 2011;14(1):61–3.

3. Galanello R, Origa R. Beta-thalassemia. Orphanet J Rare Dis. 2010;5(1):1–15.

4. Vítek L, Tiribelli C. Gilbert´s syndrome revisited. J Hepatol. 2023;79(4):1049–55.

5. Petrenko AA, Pivnik A V, Kim PP, Demidova EY, Surin VL, Abdullaev AO, et al. Coinheritanceof HbD-Punjab/β+-thalassemia (IVSI+5 G-C) in a patient with Gilbert’s syndrome. Ter Arkh. 2018;90(7):105–9.