Introduction

In a blood centre, quality assessment of the plasma / platelet components is done by various automated/semi-automated instruments. Despite advancements in automation, visual inspection of blood units remains crucial in identifying highly colored plasma units. Any plasma unit exhibiting unusually high coloration, when compared to a standard plasma unit, warrants further investigation through bilirubin testing. As an integral part of our comprehensive blood transfusion safety protocol, every donated blood unit is subjected to screening for transfusion-transmitted infectious diseases. However, a subset of seronegative donated blood units exhibits highly colored plasma with mildly elevated bilirubin levels, despite the donors being clinically asymptomatic. This phenomenon emphasizes the significance of meticulous visual inspection of donated blood components, serving as a crucial adjunct to laboratory testing, even in asymptomatic donors, to ensure the highest level of safety and quality.

Bilirubin, a yellow pigment derived from the catabolism of heme-containing proteins, most of which originates from senescent red blood cells. Under normal circumstances, serum bilirubin levels range from 0.1-1.2mg/dl, with the majority being unconjugated (0.1-1.0mg/dl).[1] Hyperbilirubinemia occurs when bilirubin levels in the systemic circulation exceed normal ranges. Elevated levels can result from disorders affecting bilirubin metabolism, hepatic dysfunction, or bile duct obstruction.[2] Notably, bilirubin levels below 2mg/dl typically do not manifest with jaundice. Jaundice, characterized by yellowish discoloration of the skin and bulbar conjunctiva, becomes apparent when the balance between bilirubin production and excretion is disrupted, usually at serum bilirubin levels ≥ 2.5mg/dl.[3]

The primary objective of this study is to determine the prevalence of mild hyperbilirubinemia among seronegative blood donors within our population. Furthermore, this study aims to discuss the underlying few factors that contribute to asymptomatic hyperbilirubinemia in blood donors.

Materials and Methods:

This study is a prospective observational study conducted in the department of Blood Transfusion Medicine at Government Coimbatore Medical College and Hospital. Donor’s blood collected from 1st July 2024 to 31st January 2025 was analysed. Totally 8454 units collected and are tested for Transfusion Transmitted Infection Screening viz: HIV, HBV, HCV, Syphilis and Malaria. The Blood donors were selected as per the Guidelines G.S.R 166(E), Ministry of Health and Family Welfare (MoHFW), Government of India, dated 11th March 2020.[4] In our blood centre, visual inspection of blood components is a routine protocol. After donation, during component preparation, a highly yellow coloured plasma bag was compared with another normal unit. All plasma/platelet component bags showing high-yellow coloration were included in the study were subjected to serum bilirubin level estimation to detect hyperbilirubinemia. A two sample method one from plasma bag and another from donor pilot tube sent to biochemistry laboratory for estimation of total and direct serum bilirubin. Serum bilirubin level was done on (Erba Mannheim) automated analyzer. Serum total bilirubin level of >1.2mg/dl is taken into consideration.

Results:

A total of 8454 units were collected during the seven months period. Among those units, 40 units (0.47%) were found to be icteric. The serum total bilirubin levels in these donors ranged from 1.4 to 6.2 with a mean level of 2.07 mg/dl. Serum unconjugated bilirubin levels ranged from 0.7 to 5.9 mg/dl with a mean level of 1.37 mg/dl. prevalence of unconjugated hyperbilirubinemia in asymptomatic healthy donors in our population is 0.29 %. Serum conjugated bilirubin levels ranged from 0.3 to 2.1 mg/dl with a mean level of 0.66 mg/dl. All units were from male donors only. The details are given in Table 1. All 40 units were negative for TTI screening. The elevation in serum bilirubin levels was found to be due to an increase in indirect bilirubin, predominantly unconjugated in nature. None of the donors presented with clinical jaundice or reported a history of jaundice, blood transfusion, or hepatitis-like illness.

The majority of donors was between age 18-20 years (27.5%) and 26-30 years (27.5%) had the same number of patients (n=11). The mean age was 28.07 years. The youngest was 19 years old and the oldest patient was 48 years of age.

Blood collection statistics of both in-house collection and camps are crucial for meeting blood supply needs. Table 3 data reveal that in-house collection accounts for 59.45% of total blood collection and voluntary blood donation camps contribute 40.55% to the total blood collection.

Discussion:

All the 40 donors with no signs of jaundice and transfusion transmitted infection marker were found to be negative. As per regulatory aspects, clinical sign jaundice is one of the deferral criteria for blood donors. [5] But the post-donation plasma sample with hyperbilirubinemia raises the question of its etiology and safety to the patient. In the present study, all donors with asymptomatic hyperbilirubinemia were males. Similarly, in the study done by Jeeva Priya et al., all 69 units were from male donors only.[6] The main reason for no female donors were due to high prevalence of anaemia among Indian women.

Our study revealed that 40 out of 8,454 units (0.47%) from donors exhibited icteric plasma over a seven-month period. In comparison, Jeeva Priya et al. reported a prevalence of 0.29% (69/23,501 units) over three years,[6] while Arora V et al. found a prevalence of 0.97% (27/2,734 units) over five years.[15] Notably, all icteric plasma units in these studies were discarded due to the absence of guidelines governing their clinical use.

In this study, majority of the donors (70%) showed increased levels of indirect bilirubin. The isolated unconjugated hyperbilirubinemia among the blood donors may be attributed to several factors, including mild red cell hemolysis, drugs that impair hepatic uptake of bilirubin, and Gilbert syndrome.[7] In most the individual with isolated unconjugated hyperbilirubinemia has given a history of occasional alcoholic intake. The possible mechanism, alcohol can impair the function of organic anion-transporting polypeptides, which are responsible for bilirubin uptake into hepatocytes, generates toxic byproducts acetaldehyde that can interfere with bilirubin transport and conjugation, and alcohol-induced hepatocyte injury can reduce the liver's ability to process bilirubin effectively.[8][9] Even in the absence of significant liver disease, alcohol can cause transient hepatocellular dysfunction, leading to mild hyperbilirubinemia.[10]

A study conducted by Koul et al. in Kashmir, India, investigated the prevalence of Gilbert's Syndrome among 1000 randomly selected blood donors. The researchers found that 3% of the donors had evidence of Gilbert's Syndrome, with a mean serum bilirubin level of 2.64 mg/dl.[11] Notably, all affected individuals were previously unaware of their hyperbilirubinemic state. Upon further investigation, no other causes of jaundice were identified. Gilbert’s syndrome (GS) is indeed a mild, inherited disorder that primarily affects bilirubin metabolism. The main feature of GS is an increased level of unconjugated bilirubin in the blood stream due to a deficiency of the enzyme UGT1A1, which is involved in the conjugation process of bilirubin.[12] The genetic mutation responsible for GS was identified in theUGT1A1 gene.[13] Many patients with Gilbert’s syndrome, experience more pronounced jaundice after alcohol intake due to additive impairment in bilirubin metabolism or when the body undergoes stress or any form of physiological strain. The prevalence of Gilbert's Syndrome is around 8%, male predominance has been observed in various studies and is consistent across different populations worldwide.[14]

Other reason for drug-induced hyperbilirubinemia in blood donors can indeed occur due to the ingestion of non-allopathic medicines, such as homeopathic or ayurvedic drugs.[15] Many donors often perceived this non-allopathic medicine as "natural" or "safe," leading individuals to not disclose it during pre-donation medical evaluations. However, certain herbs, formulations, or even interactions between different natural substances can cause liver dysfunction, leading to hyperbilirubinemia. Oxidative stress caused by certain compounds in herbs, toxic heavy metals or contaminants in unregulated products can be toxic to red blood cells and lead to hemolysis.[16] This issue underscores the importance of a comprehensive medication history, including all forms of alternative and traditional treatments, to ensure that any possible risks are appropriately assessed. Donors must be encouraged to fully disclose any and all medications, regardless of their origin, to avoid complications.

The majority of blood donors fell within two age groups18-20 years with 27.5% and 26-30 years with 27.5%. Both age groups had the highest number of donors, with 11 donors in each group. Adolescence and young adulthood are times when individuals are highly influenced by their peer group.[17] The desire to fit in, feel accepted, and gain social validation can lead young people to experiment with substances like alcohol and illicit drugs. Other reasons like easy accessibility and affordability of alcohol makes it accessible even to students and young people from lower-income backgrounds, mental stress may resort to alcohol and illicit drugs to manage their emotion, influence of media and movies portraying it as part of the lifestyle, lack of awareness and education about the risks of alcohol abuse.

Individuals from lower socio-economic groups face multiple risk factors like environmental, nutritional, and healthcare related that may contribute to the increased incidence of isolated indirect hyperbilirubinemia. They may live in areas with high environmental pollution or have occupations that expose them to toxins. Such exposure can lead to oxidative stress and damage to red blood cells leading to increased hemolysis.[16] Poor diet and malnutrition can lead to deficiencies in nutrients such as vitamins B12 and folic acid, which are essential for normal liver function. These nutrients deficiencies can impair the liver's ability to process and conjugate bilirubin, leading to an increase in unconjugated bilirubin level.[2][18]

In our study, isolated hyperbilirubinemia is more prevalent among replacement blood donors, likely due to several factors. The probable reason would be infrequent health monitoring, undiagnosed underlying conditions such as Gilbert's Syndrome, and increased physiological stress associated with blood donation.[19] On the other hand, voluntary blood donors tend to undergo more regular health evaluations and may be in better overall health, reducing the likelihood of complications like hyperbilirubinemia. Proper donor selection and medical examination of blood donors could help identify these risks and prevent potential issues.

Conclusion:

In our study conclusion, the current rules and regulations prohibit the use of blood components if the plasma color appears abnormal. However, various studies have suggested that recipients of blood with highly coloured plasma do not experience any adverse effects. Until sufficient evidence confirms the safety of transfusions involving blood with abnormally coloured plasma, our institution will continue the policy of discarding blood components with hyperbilirubinemia. Additionally, in the absence of underlying disease, the reason for discarding blood may be classified as "isolated unconjugated hyperbilirubinemia," and the donor should be referred for medical / medico gastroenterology department for follow-up. The department of social preventive medicine continues to re-emphasize the risks of adopting harmful lifestyle changes through education and awareness, particularly among adolescents and young adults. This ongoing effort aims to encourage the younger population to adopt healthier lifestyles. Finally, the regulatory authority overseeing blood centres should determine whether serum bilirubin levels, with a defined cutoff limit, should be included in the routine testing of all blood components. A comprehensive re-evaluation of existing national policies and regulations is strongly recommended.

Acknowledgements:

We acknowledge the work done by laboratory technician of blood centre and biochemistry of Government Coimbatore Medical College Hospital.

References:

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