Paramount Definition of Reference Interval for Biochemistry Parameters in Indian Sub-Population: A Healthy Adult Population Based Cross Sectional Study

Introduction

Maximum treatment modalities depend on laboratory diagnosis of various metabolic disorders by assessment of serum biochemical parameters like liver, cardiac, renal, pancreatic functions etc. Today in India, most of the normal reference intervals used for the evaluation of health status were drawn from the western population. Appropriate reference intervals for these tests are the most important elements for health evaluation, disease diagnosis, therapy monitoring, and prognosis assessment. It is therefore wise and essential to have reference values of native population to avoid false positive and false negative findings in patients evaluated for health illnesses.

According to National Accreditation Board for testing and calibration Laboratories (NABL), the exact number of clinical diagnostic laboratories in India is not known. It is estimated that there are >50,000 pathology and diagnostic labs in India. With the increasing degree of standardization of laboratory, members of the scientific community are now proposing that it is feasible to establish common reference intervals for Indians.¹

For instance, the international community has established common Biological reference intervals (BRI) through several multi-centric studies, based on the populations of five Nordic nations, seven Southeastern African countries and multiple ethnic groups from different countries respectively.^2-4 However, it is not suitable to directly apply these reference intervals to Indian population, because some parameters may vary significantly among different races.^5-14 In order to achieve this objective, there is an urgency to establish a reference interval for Indian population.

Objectives

• To determine the reference intervals of common clinical chemistry parameters in healthy adult population.

• To validate the newly established reference intervals for national applicability.

Materials and Methods

Ethical clearance: The study was approved by Padmashree Institutional Ethics committee.

Materials: After obtaining the informed consent, a cluster sampling approach was used to recruit apparently healthy, age matched individuals (considering factors such as gender, age, region, and urban-rural) from Bangalore based population of four geographical regions, such as Karnataka, Kerala, Andhra Pradesh and Tamil Nadu, constituting the south Indian population for the study. Subjects in the age range of 18 to 65 years fulfilling the following inclusion and exclusion criteria were recruited for the study.

Inclusion criteria: Healthy individuals consenting to complete a questionnaire and physical examination by physician to assess their health condition. The subjects with normotensive status (systolic pressure <140 mmHg and/or diastolic pressure < 90 mmHg), body mass index (BMI) < 25 kg/m² , alcohol consumption <30 g/day, smoking 2-3 cigarettes/day, after two weeks of medical follow-up were included in the study.

Exclusion criteria: Age <18 years, adults with common infections, hemoparasites, skin rashes, history of blood transfusion <6 months, HIV, HCV, HBV positives and HCG positive (for females), observable mental illness, disabled, chain/chronic smokers, chronic alcohol drinkers, anemic, malnourished (BMI <17.5 kg/m² ), hospitalized persons, metabolic syndrome, hormone replacement therapy, acute and chronic diseases, pregnancy and lactating women were excluded from the study as per WHO recommendations.

Study participants scheduling and specimen handling & analysis

Study participants were informed two to three days prior to sample collection. They were asked to maintain their living and dietary habits as usual but were asked avoid alcohol-intake during the last 24 hours and smoking one hour prior to blood sample collection. Fasting venous blood samples was drawn for the study in the morning after explanation of procedure. About 5 mL of venous blood samples were collected by using vacutainer system and dispensed into 5 mL gel vacutainer. Then, test tubes were placed in an icebox and were transported to Padmashree Diagnostics, Vijayanagar, Bangalore. Plain blood specimens received at the laboratory were subjected to centrifugation (1800 x g /15 mins) to separate the cellular components and the serum, processed for the analysis of routine biochemical parameters within two hours of collection.

Biochemical parameters studied

Biochemical parameters of serum, such as Fasting glucose, urea, creatinine, total cholesterol, triglycerides, HDL- C (Direct), LDL-C (Direct), VLDL-C, uric acid, total protein, albumin, T. Bilirubin, D. Bilirubin, ALT, AST, g - GT, ALP, were studied by employing appropriate IFCC recommended, published analytical methods. Further, the obtained values were constantly validated with demonstrated measure of precision and accuracy to the compliance of regulatory body by quality assurance checks.

A total number of 253 healthy study participants in the age range of 18 to 65 years were included. Out of total 253 study participants, 80 (31.6%) were males and 173 (68.4%) were females (Table 1).

Among the 253 study participants, height of 73 subjects (28.9%) was between 4´ to 5´ft and 171 subjects (67.6%) was between 5´ to 6´ft and rest of 9 subjects (3.5%) were above 6´ft (Table 2).

Among all the participants, maximum of 31.6% (80) were from Karnataka, 24.5% (62) were from Kerala, 23.7% (60) were from Andhra Pradesh and 20.2% (51) were from Tamil Nadu (Table 3).

Among all the study participants, maximum of 33.6% (86) were in the age-group of 35 to 44 years, followed by 32.8% (83) in the age group of 25 to 34 years, 25.8% (64) in the age range of 45 to 54 years and only about 3.9% (10) and 3.9% (10) were from 15 to 24 years and 55 to 64 years respectively (Table 4).

Out of 253 study participants, average body weight of 18% (47), 39% (98), 25% (64), 13% (33) and 5% (13) of the subjects was 41-50 kg, 51-60 kg, 61-70 kg, 71-80 kg and 81-90 kg respectively (Table 5).  

Analyzed biochemistry tests

Since serum specimens studied were exclusively from healthy individual population, the quantitative measures of different test parameters were grouped as certain range such as those within the ‘reference interval’ which was referred by sourced NABL accredited laboratory and is depicted in Table 6.

Sources of data analysis

After obtaining the study data by filtering and data cleanup, it was subjected to rigorous statistical analysis by using the SPSS-Version 16.0 package. Due to difficulty of the parametric technique by Gaussian adaptation through the conventional Box-Cox formula, the inter-percentile interval to estimate the reference interval was used. It is defined as an interval bounded by two percentiles of the reference distribution. It is an illogical but common convention to define the reference interval as the central 95% interval bounded by the 2.5 and 97.5 percentiles. It also consistently narrows down to 90% confidence interval of the targeted reference intervals.

Results

The demographic characteristics of the study participants are depicted in Table 7. A total of 253 apparently healthy adults consented for this study. After careful screening and biochemical analysis, further due to various preanalytical error rejection criteria, out of 257 study participants, data of 253 study participants was considered for the study process. 92.3% (234) of the study population was within the age group of 25 to 54 years. 31.6% (80) were males and 68.4% (173) were females (Table 1 & 2). The study participants were classified as Southern states and sub classified into region base, such as Kerala (n = 62), Karnataka (n = 80), Andhra Pradesh (n = 60) and Tamil Nadu (n = 51). After rigorous statistical evaluation, the data were compared with the respective reference intervals which were referred by the sourced diagnostic facility (Table 8).

To obtain a quality of reference interval, rely on the carefully screened adult healthy individual choice. Subjects with common diseases that affect levels of biochemical test outcome were excluded by specific inclusion and exclusion criteria. Summarizing the observations made on the findings from the current study may prove to be essential for evaluation of the disease, prognosis, drug response and recruitment of study participants in studies like clinical trials. However, there is lack of specific reference interval data in India for the biochemistry tests in healthy adult population. Hence, establishing population derived reference intervals would be a new avenue in Indian health care system and medical research.

In the current study, routine biochemical tests of interest were analyzed in the study participants on automated analyzers using standard IFCC methods. The equipment (Cobas Integra 400 Plus from Roche Diagnostics), reagents, controls etc. were the same. The devices were calibrated by using protein based serum calibrators and the controls and were run at normal and pathological concentrations of the analyte.

The current study outcome was obtained by measurement of healthy individuals’ chemistry test parameters directly for the establishment of reference intervals. If the relative bias between the means of measured values of reference materials obtained by the analytical systems and the assigned target values of reference materials met the desirable specification, inaccuracy of biologic variations was cautiously monitored. The attained biochemical value of interest was expressed as mean ± S.D. The overall mean ± S.D value of seventeen (17) biochemical tests were compared with reference interval and were summarized individually (Figure 12A to Q).

The study program with emphasis on obtaining the normative data of biochemistry routine test parameters from Indian healthy adult sub-populations could achieve analysis of as many as 17 test parameters. The findings from the analyzed test parameters were related to the reference interval which was referred by the sourced diagnostic laboratory facilities and an attempt was made to arrive at the consolidated reference intervals. The laboratory reporting intervals either showed minor variations or major variations (Table 7). 

Discussion

It is a hard task to establish a reference interval according to the Clinical and Laboratory Standards Institute ratified guidelines (CLSI Guidelines) in “defining, establishing, and verifying RIs in the clinical laboratory.” The process of verifiable and perform to arrive at a recognizable own reference interval to practice is far from reality in laboratory medicine. In India, most of the laboratories follow reference intervals established in the western population or refer to the values denoted in the kit brochures.^15,16

A total number of 253 study participants’ serum specimens were subjected to biochemical analysis for seventeen test parameters. Eighty subjects were males and 173 subjects were females. No significant difference in observed values between the genders was noted. In the present study, some of the analyzed test parameters like serum fasting glucose (Figure 1A), Triglycerides (Figure 1F), HDL-Chol (Figure 1G), Albumin (Figure 1I), Total Protein (Figure 1J), D. Bilirubin (Figure 1L), ALT, AST, ALP and Calcium (Figure 1M to Q), did not show much difference compared to the reference interval.

Interestingly, there was significant difference in blood urea, creatinine, uric acid, total cholesterol (Figure 1B to E), LDL-Cholesterol (Figure 1H), T. Bilirubin (Figure 1K) and GGT (Figure 1Q) levels against the referred laboratory reference intervals. This remarkable difference underscores the need for establishment of Indian sub-population based reference intervals for use in medical care and medical research (Table 9).

Summary

Health is wealth for an individual to garner socioeconomically, but conceptually in the same country like India with multi-diversity population, the quality of life of individuals remarkably defers by relative and not an absolute state. Hence, the condition of individuals must be related to or compared with reference data. There have been very few published papers on South Indian population RIs for biochemical analytes.^15–22 However, a considerable effort has been made by Ashavaid TF et al.,^23,24 to arrive at derived reference intervals for Indian sub-population. Therefore, the current study made an effort to compare the RIs obtained with those sourced from NABL accredited laboratory (Table 8).

Limitation of the current study

The limitation of this study could be the sample size (n=253), which is quiet trifling with India being culturally and customarily multi diverse. It is very difficult to select a reference group that represents the entire population.

Salient features of the study outcome

• The current study produced reference intervals of various biochemical parameters and these were compared to validated reference intervals.

• By and large, many biochemical test RIs were closer to sourced NABL accredited laboratory reference intervals.

• The present study demonstrated reference intervals for seven parameters (Urea, Creatinine, Uric acid, T. Cholesterol, LDL-Cholesterol, T. Bilirubin and g-GT) which were remarkably diverse from sourced RIs.

• The pattern emerging from these studies could be a deciding factor for revising the reference values and the derived RIs depicts remarkably lower reference limit than the available reference interval except for LDL-Cholesterol (Table 8 & Figure 12 H).

• Hence, it is an appropriate diagnostic reference for clinical decision-making which may help in reducing the diagnostic dilemma of clinicians.

Conclusion

Among 17 clinical chemistry parameters tested, only LDL-Cholesterol level was 37% above the mean reference limit. The lipid fraction calls for immediate attention to revise the reference limit. However, it is no doubt the result by and large could be categorized as normal but the bad cholesterol would be hostile for the Indian population as they may be more prone to CAD/CVD. Rest of the parameters were harmonized with reference interval. Therefore, the study outcome emphasizes that firm adherence to the reference values of non-representative population could lead to inappropriate diagnosis and treatment of patients. The BRI was established in this study by constituting apparently healthy South Indian population. It may be a stepping platform and practical role in the determination of BRI of clinical-chemistry analytes in the future for pan India.

Conflicts of interest

Authors of the study have no financial interest nor received any financial support from the companies manufacturing reagents and instruments.

Acknowledgement

We thank Rajiv Gandhi University of Health Sciences (RGUHS), Padmashree diagnostics, Padmashree Institute of Medical lab technology (PIMLT) and Padmashree Institute of Clinical Research (PICR) for the support in completing the project.