Original article / research
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Neonatal Hyperbilirubinemia-Evaluation of Total Calcium, Ionised Calcium, Magnesium, Lactate and Electrolytes |
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Vani Axita Chandrakant, Vidya S Patil, Deepti G Ingleshwar, Vijayetha P Patil, Shilpasree A S 1. Associate Professor, Department of Biochemistry, SDM College of Medical Sciences and Hospital, Dharwad, Karnataka, India. 2. Professor and Head, Department of Biochemistry, SDM College of Medical Sciences and Hospital, Dharwad, Karnataka, India. 3. Assistant Professor, Department of Biochemistry, SDM College of Medical Sciences and Hospital, Dharwad, Karnataka, India. 4. Associate Professor, Department of Biochemistry, SDM College of Medical Sciences and Hospital, Dharwad, Karnataka, India. 5. Assistant Professor, Department of Biochemistry SDM College of Medical Sciences and Hospital Dharwad, Karnataka, India. |
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Correspondence
Address : Dr. Vani Axita Chandrakant, Associate Professor, Department of Biochemistry, SDM College of Medical Sciences and Hospital, Sattur, Dharwad-580009, Karnataka, India. E-mail: draxitacvani@gmail.com |
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ABSTRACT | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
: The Extra-Pulmonary Tuberculosis (EPTB) is challenging due to the pauci-bacillary nature of the disease and limited tests available for early diagnosis. It is the most common extrapulmonary manifestation of tuberculosis. The samples from extrapulmonary infection will have low bacterial count as compared with sputum specimens. It is also necessary to rule out other causes of granulomatous inflammation on Fine Needle Aspiration Cytology (FNAC) and confirmation by Ziehl Neelsen (Z-N) stain and Cartridge Based Nucleic Acid Amplification Test (CBNAAT). In December 2010, World Health Organisation (WHO) recommended GeneXpert/ CBNAAT to be used as the initial diagnostic test in suspected EPTB cases. Aim: To Compare FNAC with CBNAAT in the diagnosis of suspected tubercular lymphadenopathy in a tertiary care centre and to know the importance of CBNAAT in the modern era. Materials and Methods: This was a hospital based prospective study carried out over a period of 12 months (July 2019 to June 2020) in the Department of Pathology and Microbiology, ESIC Medical College. All presumptive cases of tubercular lymphadenopathy and purulent aspirates from the lymph nodes of various sites were included in the study. Smears were made after FNA and stained with Haematoxylin and Eosin (H & E) stain and Z-N stain and sample was also processed for CBNAAT in all cases of lymphadenopathy. Statistics was done using SPSS software version 20.00. Results:The total number of cases with presumptive tubercular lymphadenitis was 119. Majority of the aspirates are from jugular lymph nodal and cervical swellings 64.7% (77/119). FNAC has detected tuberculosis in 20/119 (16.80%) cases and Z-N stain detected AFB in 6/119 (5.04%) cases. CBNAAT has detected 23/119 (23.52%) cases, among them 12.60 % of cases (15/119) which were not detected by FNA. Diagnostic Performance of CBNAAT versus FNAC (28/119) showed sensitivity, specificity, negative predictive value and positive predictive value 80%, 85.8%, 65% and 45.5%. Conclusion:CBNAAT can be added with FNAC to get more specific results. CBNAAT is less sensitive for blood stained samples than purulent samples and hence FNA still remains as the cheapest and first line test to diagnose in cases suspected of tubercular lymphadenoapathy | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Keywords : Biochemical disturbances, Jaundice, Unconjugated bilirubin | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
INTRODUCTION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Bilirubin is derived from breakdown of senescent Red Blood Cells (RBCs) in the reticuloendothelial system. This bilirubin is transported to liver for conjugation and excretion. Any defect in production, transport or degradation of bilirubin may result in accumulation of bilirubin in the skin and mucus membranes resulting in jaundice. A total serum bilirubin level above 5 mg/dL is defined as neonatal hyperbilirubinemia. In the first week of life around 60% of term and 80% of preterm babies develop jaundice, and at 1 month about 10% of breastfed babies are still jaundiced (1). The risk factors for developing hyperbilirubinemia in newborn babies include maternal factors like blood type RH or ABO incompatibility,drugs like oxytocin, diazepam, ethnicity (native American, Asian), breast feeding, maternal illnesses like gestational diabetes and neonatal factors like birth trauma (instrumented delivery, cephalohematoma, cutaneous bruising), drugs (sulfisoxazole acetyl with erythromycin, chloramphenicol, ethylsuccinate), excessive weight loss after birth, infrequent feedings, infections (TORCH), polycythemia, male gender, prematurity and previous sibling with hyperbilirubinemia (2). Bilirubin has a neurotoxic effect. When the levels of bilirubin =25 mg/dL it can cross the blood brain barrier and via apoptosis and necrosis it can produce irreversible damage. Choudhary and colleagues noted that among babies with neonatal hyperbilirubinemia ABO incompatibility was twice as common as Rh incompatibility (3). A study found that from the third postnatal day till two weeks after birth, the concentration of ionized calcium in a retrospective selected reference population of healthy full-term and pre-term neonates significantly exceeded the upper 95% reference limit for adults (4). Magnesium is involved in maintaining bone health, carbohydrate metabolism, regulation of blood pressure, muscle contraction, energy transport and also serves a cytoprotective role (5). Since, magnesium is required for more than 300 biochemical reactions in the body, magnesium deficiency can cause various disturbances including hypocalcaemia, hypokalaemia and cardiac and neurological manifestations. Prakash CB and coworkers studied magnesium levels in 56 newborn and found that no appreciable changes occurred in the first week of life, cord blood values were somewhat higher than serum levels, no differences noted in breast as opposed to bottle feeding and no significant differences between venous and capillary blood (6). Lactate can be produced by several mechanism. It is the end product of anaerobic glycolysis which usually indicates inadequate blood perfusion. It is nowadays being considered as a marker of strained cellular metabolism (7). In conditions like sepsis or trauma, septic shock, blood loss, volume depletion, and systemic inflammatory syndrome, lactate levels can alter. Normally lactate is cleared by the liver and a small amount may be cleared by the kidneys. Any liver dysfunction can result in increased synthesis or decreased elimination. Lactate levels in babies with unconjugated hyperbilirubinemia was evaluated to try to establish a relation with indirect bilirubin. Moderate to high level of bilirubin in neonates may cause harm to developing Central Nervous System (CNS), particularly to cerebellum and basal ganglia causing severe motor symptoms and cerebral palsy (8). | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MATERIAL AND METHODS | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
A two year retrospective analysis of data (January 2015 to December 2016) was carried out at SDM College of Medical Sciences and Hospital, Dharwad, Karnataka, India, in the Department of Biochemistry. Ethical clearance has been obtained from the Institutional Ethical Committee. A total of 82 newborn babies with unconjugated hyperbilirubinemia were included in the study and 82 age and sex matched healthy newborn babies were selected as control group. All birth details and results of biochemical investigations were recorded in a prestructured proforma. Babies with conjugated hyperbilirubinemia, inborn errors of metabolism, asphyxia, congenital defects, age >2weeks, congenital hypothyroidism, presence of history of drug exposure and sepsis were excluded from the study. Biochemical investigations like serum bilirubin, lactate levels, total calcium, magnesium, urea, and creatinine which were analysed by fully automated Siemens autoanalyser and ionised calcium by ABG analyser was noted down. Statistical Analysis All data was collected in a prestructured proforma containing all the variables of interest. Data was cross tabulated and processed. SPSS software was used for statistical analysis (version 20). The test statistics used for data analysis were descriptive statistics, Pearson’s correlation, Student’s ‘t’ test with the level of significance set at 0.05 and a p-value of <0.05 was considered significant. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RESULTS | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
The sexwise distribution of controls and cases along with the demographic data of the study population is presented in (Table/Fig 1). The values indicate a higher incidence of unconjugated hyperbilirubinemia in male babies (57.3%), babies born by caesarean section (65.8%) and in babies born at <35 weeks of gestation. Levels of urea, phosphorous, sodium, potassium and chloride were significantly higher in hyperbilirubinemia babies. Level of total calcium was significantly lower. Ionised calcium and creatinine were found to be decreased and magnesium was increased in babies with indirect hyperbilirubinemia but results are not significant (Table/Fig 2). (Table/Fig 3) indicates that in jaundiced newborn the values of total bilirubin correlated negatively with ionized calcium and magnesium. In healthy neonates total bilirubin correlated negatively with magnesium, total calcium and ionized calcium. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
DISCUSSION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
In healthy term newborn, physiological jaundice follows a typical pattern where total serum bilirubin peaks at 5 to 6 mg/dL on third to fourth day of life and then slowly decreases during first week of life (1). According to All India Institute of Medical Sciences NICU protocols 2007, in term neonate if Total Serum Bilirubin (TSB) concentrations on the first day of life >5 mg/dL, on 2nd day of life >10 mg/dL or > 13 mg/dL thereafter it is non-physiological and if TSB is > 17 mg/dL it should be regarded as pathological and investigated with possible intervention like phototherapy (9). Bilirubin Toxicity Because of its affinity to membrane phospholipids, bilirubin inhibits uptake of tyrosine affecting synaptic transmission and inhibits N-methyl-D-aspartate (NMDA) receptor ion channels thus interfering with neuroexcitatory signals and nerve impulse conduction, particularly of auditory nerve. Warren BK has explained the selective toxicity of bilirubin to neonatal brain due to the immature blood brain barrier (BBB) of newborn,qualitative and quantitative differences in lipid composition of neonatal brain and inhibition of NAD dependant dehydrogenases by bilirubin, causing bilirubin to pass more readily into brain of neonates (10). Although, increased free bilirubin may be predictive of kernicterus, in some infants other factors like low APGAR score, hypothermia, hypoglycemia, sepsis, acidosis, hypoxemia and hypercarbia may make the BBB more permeable to low levels of free bilirubin (11). Bilirubin also serves as an antioxidant. Both conjugated and unconjugated bilirubin inhibits lipid peroxidation while biliverdin and bilirubin mediate a cytoprotective role through hemeoxygenase. Bilirubin also inhibits ion exchange and water transport in renal cells causing neuronal swelling in kernicterus. Bilirubin inhibits mitochondrial enzymes and interferes with DNA synthesis, produces strand breakage and inhibits phosphorylation and protein synthesis (12). Bilirubin, Gestational Age and Birth Weight Compared to term infants, late preterm infants are physiologically immature, and pose limited compensatory responses to extra uterine environment. Out of 82, 43 babies (52.43%) were less than 35 weeks of gestation and the mean birth weight was 2.1 kg of jaundiced babies and 3.18 kg of control group. These findings are similar to another study by Mahmud H and coworkers, where serum bilirubin was found to be negatively correlated with gestational age and birth weight, where preterm and low birth weight babies developed severe and very severe jaundice compared to term babies (p<0.001) (13). Another worker also found that late preterm infants (gestational age 34-36 weeks) have a higher incidence of hyperbilirubinemia along with respiratory distress, temperature instability, feeding difficulties, hypoglycemia, apnea and late onset sepsis (14). William AK et al., also noted higher morbidity and mortality in late preterm infants as diagnosed by hypoglycemia, temperature instability, respiratory distress, apnoea, jaundice and feeding difficulties. Risk factors for hospital re-admission or morbidity included being first child, being breast fed at discharge, having a mother with labor complications and being of Asian/ Pacific island descent (15). Bilirubin and Magnesium After calcium, magnesium is the second most important divalent cation in serum, with approximately 1% of total magnesium being extracellular. Magnesium ion is an important antagonistic regulator of the NMDA receptor ion channel (16). It protects central nervous system against hypoxia and exerts neuroprotective effect by blocking excitotoxic and NMDA receptor mediated neuronal injury. Magnesium physiologically acts against or compensates for the neurotoxic effects of bilirubin. 25% of total serum magnesium is bound to albumin and 8% to globulins. In this study, it was noted that magnesium levels are non-significantly increased (p=0.280) in babies with indirect hyperbilirubinemia and a negative correlation was observed between total bilirubin and magnesium (r = -0.111). Abdel-Azeem ME et al., noted that neonates with indirect hyperbilirubinemia had higher magnesium levels. In addition they found higher copper and lower zinc values than healthy babies which were not related to their maternal serum values (17). Similarly, Imani M et al., noted that magnesium levels are elevated in babies with moderate and severe hyperbilirubinemia. They noted a positive correlation between magnesium levels and bilirubin before and after phototherapy (18). Contrary to this study, Mohamed SE et al., noted a positive relation between magnesium and bilirubin and that phototherapy decreases serum magnesium and bilirubin levels (19). Sarici and colleagues demonstrated a positive correlation between severity of hyperbilirunemia and Ionised Magnesium (IMg) and significantly higher plasma IMg levels in severe hyperbilirubinemia group than group with moderate hyperbilirubinemia. This elevation in plasma IMg may be observed due to extracellular movement of intracellular magnesium resulting from cellular injury of neurons and red blood cells (20). Bilirubin and Calcium Direct measurement of ionsed calcium rather than total calcium, is the method of choice when accurate knowledge of active concentration of calcium is required (21). Capillary blood sample may be used but ionised calcium in capillary blood may be slightly higher than in arterial blood. Light induced hypocalcemia (following phototherapy) results from increased calcium uptake by bone when inhibitory effect of melatonin declines after pineal inhibition by transcranial illumination (22). In this study mean total calcium in the control group was 9.57 mg/dL and 8.66 mg/dL in case group. The mean ionised calcium level was 5.15 mmol/L in control group and 4.95 mmol/L in case group. Total bilirubin correlated negatively with total and ionized calcium in control babies but in jaundiced babies total bilirubin correlated positively with total calcium and negatively with ionized calcium. These findings are similar to a study by Reddy AT et al., who found higher incidence of hypocalcaemia and hyponatremia in Low Birth Weight (LBW) babies and preterm neonates with jaundice (23). Similarly, Dushyant R et al., found hypocalcaemia more common in preterm and term newborn having higher TSB (24). Statistically significant difference was noted in total calcium levels between TSB group15.1-20.0 mg/dL and TSB group 20.1-30.0 mg/dL. Preterm babies developed more hypocalcaemia symptoms than term babies. Hosny and coworkers found plasma bilirubin and plasma IMg levels to be significantly higher in hemolytic and non-hemolytic jaundiced cases. Ionised calcium was significantly higher in hemolytic cases and not significant in non hemolytic cases in each group of full term, preterm and LBW babies (22). Amit J et al., found that after exchange transfusion total bilirubin decreased, but levels of calcium and phosphorous remained unchanged in jaundiced newborn (25). Hypomagnesemia also causes a defect in synthesis and release of Parathyroid Hormone (PTH) and end organ resistance to PTH leading to hypocalcaemia. Magnesium therapy appears to have an important role in rectifying not only hypomagnes aemia but also hypocalcaemia. Bernard LS et al., have concluded that prophylactic administration of continuous intravenous calcium gluconate at 35 mg/kg/24hr limits the postnatal depression of serum calciumin LBW infants during first 24 hours of life (26). Findings of several studies suggest that hypermagnesemia and hypocalcaemia may be critical factors in development of tissue injury (27),(28),(29),(30). Bilirubin and Other Electrolytes In present study electrolytes sodium, potassium and chloride levels were significantly elevated in babies with indirect hyperbilirubinemia. These findings are consistent with those of another study (31). But in the study by Reddy and colleagues, incidence of potassium and chloride changes following phototherapy was nonsignificant irrespective of gestational age, birth weight and duration of phototherapy (23). Similarly, a study by Amit J and coworkers found that after exchange transfusion total hemoglobin and bilirubin decreased, but levels of urea, potassium remained unchanged in jaundiced newborn (25). Sunil KP and Yadav showed that premature and LBW babies are at a higher risk of developing hyponatremia if they receive phototherapy (32). It was found that levels of urea were significantly higher in the case group. But difference in creatinine levels between case and control group was non significant. Bilirubin and Lactate In clinical practice elevated levels of lactate are indicative of any form of shock or tissue hypoperfusion. Lactate is elevated in a variety of situations which include sepsis and septic shock, cardiogenic, obstructive and hemorrhagic shock, cardiac arrest, trauma, seizure, excessive muscle activity, regional ischemia, burns and smoke inhalation, diabetic ketoacidosis, thiamine deficiency, malignancy, liver dysfunction, inborn errors of metabolism, drugs like metformin and alcohol. In this study lactate levels were found to be slightly higher than the reference range in babies with indirect hyperbilirubinemia. However, this was not compared with healthy control group as it is retrospective analysis of data. LIMITATION A wide variety of biochemical disturbances are seen in babies with indirect hyperbilirubinemia but the exact mechanisms leading to these disturbances need to be elucidated. As this is a retrospective analysis of hospital data, lactate levels could not be determined in control babies, which is the limiting factor of current study. The study can be improvised by studying changes in the levels of biochemical parameters before and after initiation of appropriate treatments. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CONCLUSION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Unconjugated hyperbilirubinemia is a common condition seen in newborn babies. It was noted that total calcium was decreased significantly but changes in levels of ionized calcium and magnesium were not significant in babies with indirect hyperbilirubinemia. Further, studies can be carried out on larger population samples to firmly establish the relationship between biochemical markers and neonatal hyperbilirubinemia. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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TABLES AND FIGURES | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||