Afshan Bibi  ( Department of Chemical Pathology and Endocrinology, Armed Forces Institute of Pathology, Rawalpindi, Pakistan. )
Zujaja Hina Haroon  ( Department of Chemical Pathology and Endocrinology, Armed Forces Institute of Pathology, Rawalpindi, Pakistan. )
Amna Shujaat  ( Department of Chemical Pathology and Endocrinology, Armed Forces Institute of Pathology, Rawalpindi, Pakistan. )
Muhammad Aamir  ( Department of Chemical Pathology and Endocrinology, Armed Forces Institute of Pathology, Rawalpindi, Pakistan )
Sobia Irum  ( Department of Chemical Pathology and Endocrinology, Armed Forces Institute of Pathology, Rawalpindi, Pakistan. )
Syed Raza Jaffar  ( Department of Chemical Pathology and Endocrinology, Armed Forces Institute of Pathology, Rawalpindi, Pakistan. )

Objective: To determine the reference interval of biotinidase activity in healthy neonates.

 

Method: The cross-sectional study was conducted at the Department of Chemical Pathology and Endocrinology, Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from May to November 2019, and comprised blood samples collected from healthy neonates aged 2-6 days. The samples were collected on filter paper and analysed on genetic screening processor based on dissociation-enhanced lanthanide flouroimmunoassay. Data was analysed using SPSS 21.

 

Results: Of the 120 dried blood spot specimens, 81(67.5%) were from male babies and 39(32.5%) from female babies. Reference interval for biotinidase activity, based on 2.5th and 97.5th percentiles, was from 3.0 to 11.0 nmol/ml/min.

 

Conclusion: Screening of newborns for biotinidase deficiency is crucial to prevent irreversible neurological damage.

 

Keywords: Biotinidase reference interval, Biotinidase deficiency, Newborn screening in Pakistan. (JPMA 72: 97; 2022)

 

DOI: https://doi.org/10.47391/JPMA.2167

 

Introduction

 

Among the genetic disorders, biotinidase deficiency (BD) is considered the most rewarding metabolic disorder to treat.¹ BD is not common worldwide, but in countries like Saudi Arabia,² Turkey³ and Iran,⁴ where the rate of consanguineous marriages is very high, this disorder is very common. One out of 60,000 children worldwide are born with BD.⁵ BD is reported recently in multiple case reports from Pakistan^6-8 and it has also been reported in Pakistani children residing in other countries.⁹ A study in Karachi reported 33 cases of confirmed BD out of 113 cases reported with signs and symptoms of BD over a 10-year period from a single centre.¹⁰ This shows that BD is very much present in Pakistan, but, due to lack of diagnostic facilities and awareness of disease, the disease is rarely diagnosed.

BD is an inherited, autosomal recessive disorder of biotin recycling, also referred to as multiple carboxylase deficiency.¹¹ Biotin is an essential vitamin that serves as a coenzyme for four carboxylases, including propionyl-Coenzyme A (COA) carboxylase, 3-methylcrotonyl-COA carboxylase, pyruvate carboxylase, and acetyl-COA carboxylase, in humans. These enzymes play important roles in carbohydrate, amino acid and fatty acid metabolism. The major cause of this disease is absence or abnormal activity of a cytosolic enzyme biotinidase. Newborns having BD may or may not show symptoms at the time of birth. Based on the level of activity of biotinidase enzyme in the serum of the patients, the disease is classified as partial deficiency 10-30% enzyme activity, and profound deficiency 0-10% enzyme activity. The condition is easily treatable by biotin supplements, but, if not diagnosed timely, it can lead to severe consequences, like irreversible neurological damage, sensorineural deafness, developmental delay and seizures.¹²

The National Institute of Population Studies in 2013 reported that 48.5% of all marriages in Pakistan occur between first cousins and 7.9% between second cousins.¹³ The more the consanguinity, the more is the risk of having genetic disorders.¹⁴ The incidence of inherited metabolic disorders varies from country to country, and, compared to developed countries, there is predicted ten-fold higher incidence of inherited metabolic disorders (IMDs) in Pakistani children.¹⁵ In Pakistan, infant mortality rate is 62 per 1000 live births.¹⁶ It is also a fact that IMDs did not gain much importance and have been ignored by both the private sector as well as the government.

Biotinidase testing is part of newborn screening programme in most developed countries. Determining reference values for biotinidase in healthy neonates bear paramount importance for the launching of a neonatal screening programme in Pakistan. Early and prompt diagnosis can prevent irreversible neurological damage and even death of the neonates. The current study was planned to determine the reference interval of biotinidase activity in healthy Pakistani neonates.

 

Materials and Methods

 

The cross-sectional study was conducted at the Department of Chemical Pathology and Endocrinology, Armed Forces Institute of Pathology (AFIP), Rawalpindi, Pakistan, from May to November 2019. After approval from the institutional ethics review, the sample size was calculated in line with Clinical and Laboratory Standards Institute (CLSI) guidelines.¹⁷ The sample was raised using non-probability convenience sampling technique. committee After written informed consent from the parents, blood samples were taken from the Paediatrics Department of the Pakistan Emirates Military Hospital (PEMH), Rawalpindi. The samples were taken from healthy neonates aged 2-6 days. . Neonates with history of sepsis or IMDs or on some management strategy, like dietary restrictions or enzyme replacement, and premature neonates as well as those who had received blood transfusions were excluded.

Demographic characteristics, family history, biochemical findings and results of biotinidase assay were recorded. Preliminary biochemical tests included complete blood count (CBC), plasma ammonia, lactate, plasma glucose, arterial blood gas analysis, urine for non-glucose reducing substances, and liver functions tests (LFTs). These tests were done to exclude sick newborns from the study. Only newborns who had normal results for all these tests were included in the study. Dried blood spot (DBS) was collected on Guthrie cards for biotinidase estimation through heel prick test. DBS samples were transported in special plastic bags protected from direct sunlight and humidity. Biotinidase enzyme analysis was carried out on a fully automated analyser with loaded microtitration plates containing DBS (Genetic Screening Processor 2021, Perkin Elmer). The sample was extracted from DBS by the extraction solution present in the reagents as a first step of the assay. Biotinidase assay (Kit product No. 3307-0010) was used containing biotinidase substrate (Europium- labelled biotin) reagent, biotinidase streptavidin reagent, assay buffer and anti-streptavidin immunoglobulin-G (IgG) microtitration strips. Biotinidase assay was based on an enzyme reaction with a solid phase time-resolved immunofluorescence assay. The amide bond in europium-labelled biotin was cleaved by the biotinidase enzyme present in the sample. The enzyme reaction was stopped by adding streptavidin and the resultant streptavidin-biotin complexes were captured by solid phase monoclonal antibodies. Dissociation-enhanced lanthanide flouro-immunoassay (DELFIA) inducer dissociated the molecules into the solution where the europium fluorescence was measured. The measured fluorescence was inversely proportional to the biotinidase activity in the sample. For the biotinidase assay, six standards were used for every analytical run. In every plate, medium and high levels of standards (six for each) were measured. Intra- and inter-assay coefficient of variation (CV) was 3.3% and 7.2% respectively. A positive abnormal and normal control was included with each batch of samples. Data was analysed using SPSS 21. Kolmogorov Smirnov test was applied where necessary. P<0.05 was considered significant. The 2.5th and 97.5th percentiles were computed using the formula 0.025(n+1) and 0.975(n+1), which corresponded to rank number 3 and 118 respectively with 90% confidence interval (CI).

 

Results

 

Of the 120 DBS specimens, 81(67.5%) were from male babies and 39(32.5%) from female babies. Histogram showed non-parametric distribution (p<0.05) (Figure).

 

 

Data was arranged in ascending order and rank number was allotted (Table 1).

 

 

Reference interval of biotinidase, established on the basis of 2.5th and 97.5th percentiles, was from 3.0 to 11.0nmol/ml/min (Table 2).

 

 

 

Discussion

 

Health-associated reference values for every laboratory analyte are universally required for the interpretation of medical laboratory results, as a decision-making process. The current study determined reference values for biotinidase activity (from 3.0 to 11.0 nmol/ml/min) in Pakistani neonates so that newborn screening for BD can be started in Pakistan.

A mass screening study was conducted in Belgium and Turkish populations in 2002.¹⁸ Belgian population’s biotinidase reference interval was significantly higher than the Turkish population. Reference interval for biotinidase was found to be from 4.85 to 5.63nmol/ml/min for Belgian population and from 4.16 to 5.86 for Turkish population.¹⁸

A study in Iran reported biotinidase activity values from 3.81 to 8.35nmol/ml/min, identifying 8 patients with BD out of 47 children with neurological signs. Two mutations were detected, c.98-104del7ins3 and p. Arg79Cys, in 5 patients with profound BD, and one p. Asp444His mutation in 3 patients with partial BD.⁴

A study in Brazil reported biotinidase reference interval from 5.2 to 9.5nmol/ml/min.¹⁹ A study from India reported biotinidase activity levels from 4.52 to 9.14nmol/ml/min.²⁰

Biotinidase activity levels show slight variation among different populations. This variability depends on ethnicity, dietary patterns, genetic makeup and traditions of interfamily marriages. The reference interval in the current study showed more resemblance with Iran, India and Turkey^4,18,20 (Table 3). This is because of the of similarities in ethnicity, dietary habits and same traditions of consanguineous marriages in these communities.

 

 

Newborn screening should be established as an important tool to screen for inborn errors of metabolism at the time of birth. However, the current state of newborn screening is not very good in Pakistan. The main challenges in establishing this programme have been the lack of funds, less awareness in public, inadequate manpower and support services. Early diagnosis may save a number of children from getting disabled. In fact, pre-symptomatic evaluation of these conditions through newborn screening will definitely minimise the irreversible, life-threatening side effects of the disease. The present study put forward reference interval of biotinidase in Pakistani neonates, but many such studies are required to be done in this regard so as to establish the actual burden of BD in the country, its frequency and strategies to cope with this situation.

Appropriate measures are being taken by other neighbouring countries, like Iran and India^4,20 for progress in this regard. Diagnostic facilities are being established and continuous improvements are being done to screen neonates at the appropriate time and to initiate treatment, if required, to stop the development of irreversible neurological damage.

 

Conclusion

 

The reference interval of biotinidase for disease-free newborns was found to range from 3.0 to 11.0nmol/ml/min. In Pakistan, the BD prevalence is not known, but is expected to be high due to high rate of consanguineous marriages.

 

Disclaimer: None.

Conflict of Interest: One of the co-authors heads the institutional ethics review board.

Source of Funding: None.

 

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