Year :
2022
| Month :
October
| Volume :
16
| Issue :
10
| Page :
BC10 - BC13
Full Version
Reference Interval of Serum Cystatin C in Dravidian Population Subset of South Asian Ethnic Groups: An Observational Cross-sectional Study
Published: October 1, 2022 | DOI: https://doi.org/10.7860/JCDR/2022/58894.16965
Shajee Sivasankaran Nair, K Geetha Damodaran, Sestin Sebastian
1. Associate professor, Department of Biochemistry, Government Medical College, Manjeri, Kerala, India.
2. Professor, Department of Biochemistry, Government Medical College, Thrissur, Kerala, India.
3. Senior Resident, Department of Biochemistry, Sree Narayana Institute of Medical Sciences, Chalakka, Kerala, India.
Correspondence Address :
Dr. Sestin Sebastian,
Senior Resident, Department of Biochemistry, Sree Narayana Institute of Medical Sciences, Chalakka, Kerala, India.
E-mail: sestinsebastian19@gmail.com
Abstract
Introduction: Serum Cystatin C (Cys C) facilitates to detect mild renal dysfunction and overall risk of death in older patients with chronic renal diseases. The requirement for creating a particular reference interval is mandatory for such renal diseases in South Asian population since the value of serum creatinine varies largely with age, gender and population/ethnicity and data of same is short for South Asian population.
Aim: To determine the reference interval of serum Cystatin C amongst the Dravidian population subset of South Asian ethnic groups.
Materials and Methods: This observational cross-sectional study was conducted in Department of Biochemistry and Transfusion Medicine at Government Medical College, Thrissur, Kerala, India, from July 2020 to January 2021 among on 235 healthy adults (20-60 years). A 4 mL of blood was drawn by aseptic precautions and serum was separated within three hours of blood collection. Serum Cys C was assayed by latex enhanced immunoturbidimetric cystatin C assay using a calibrator traceable to international standard European Specific Protein Reference Material (ERM DA-471) developed by the International Federation of Clinical Chemistry (IFCC) and Laboratory Medicine. Serum Creatinine (sCr) by Jaffe method in a fully automatic clinical chemistry analyser was assessed. Categorical variables are presented using frequency (percentage) while continuous variables are summarised with an interpercentile range. The Kolmogorov-Smirnov test was used to assess the normality of the distribution of continuous variables.
Results: Total of 235 subjects (20-60 years of age with 17 males and 218 females) were included in the study. The non parametric reference intervals for Cys C were found to be from 0.39 to 0.79 mg% and that of sCr ranged from 0.79 to 1.2 mg%. The relationship between Cystatin C with age showed an increase in Cystatin C levels as age advances; Spearman’s rank correlation coefficient (rho=0.197). The weak correlation between Cystatin C and sCr was also observed (rho=0.37; p-value <0.0001).
Conclusion: The level of serum cystatin C (0.39 to 0.79 mg%) can be used as a diagnostic concentration reference interval of the protein that helps to recognise, standardise and establish it as a potential biomarker to detect renal disorders in South Asian ethnic population.
Keywords
Biomarkers, Contrast-induced nephropathy, Glomerular filtration rate, Renal diseases, Serum creatinine
Introduction
It has been estimated by the World Health Organisation (WHO), that approximately 85,000 patients suffer from Chronic Kidney Disease (CKD) every year across the globe, and around 17.2% of such cases are seen to be prevalent in India (1),(2). Studies have proven that the progression of kidney disease can be restrained or even prevented by early detection and establishment of appropriate treatment.
The use of estimated Glomerular Filtration Rate (eGFR) is recommended in current clinical practice guidelines for the assessment of specific renal diseases (3),(4). The most commonly used formulae comprises of the Cockcroft-Gault formula, the modification of diet in renal disease, and the Chronic Kidney Disease Epidemiology Collaboration formulae, which uses serum Creatinine (sCr) for estimating GFR (5). Such measurements of eGFR consists of numerous drawbacks, such as the incapability to distinguish early renal dysfunction owing to its low sensitivity (6). Owing to such restrictions, serum Cystatin C (Cys C) has been anticipated as an ancillary endogenous marker for renal diseases (7). Cys C is easily filtered and is entirely catabolised at the proximal tubule without the help of tubular secretion. Cystatin C being produced by all nucleated cells, is a member of the cystatin superfamily of cysteine protease inhibitors. It is a non glycosylated, low molecular weight, basic protein, consisting of 120 amino acids (8),(9),(10). There are numerous mechanised procedures that have been adopted to measure the protein like the Particle Enhanced Turbidimetric Immunoassay (PETIA), an in-house latex PETIA and a latex Particle Enhanced Nephelometric Immunoassay (PENIA) (11),(12),(13).
The assay of serum Cystatin C has several uses in clinical medicine wherein it proves to be a better marker than sCr to detect early stages of Contrast-induced Nephropathy (CIN) (14),(15). Serum Cystatin C can detect mild renal dysfunction and overall risk of death in older patients and higher mortality in cases of acute coronary syndrome (16),(17),(18). Hence, the present study was planned to find the reference interval of serum Cystatin C amongst the Dravidian population subset of South Asian ethnic groups.
Material and Methods
This observational cross-sectional study was conducted in Department of Biochemistry and Transfusion Medicine at Government Medical College, Thrissur, Kerala, India, from July 2020 to January 2021. The approval was obtained from the Institutional Ethical Committee {letter no. B6-155/2019/MCTCR(28); dated- 02/03/2019}. A total of 252 healthy adults between the age group 20 to 60 years were selected after getting informed consent from volunteers of blood donation registered in the Transfusion Medicine Department.
Inclusion criteria: Healthy male and female patients aged between 20-60 years non smokers and not on steroids therapy, or any medications were included in the study.
Exclusion criteria: Subjects with Thyroid Stimulating Hormone (TSH) values outside the range of 0.4 to 4.5 mIU/L and with C-Reactive Protein (CRP) levels more than 10 mg% (19) were excluded from
the study.
Sample size: For the present study, sample size was set as 120 as per National Committee for Clinical Laboratory Standards (NCCLS) for finding out the reference interval of a biomarker (20). An additional 132 subjects were included to accommodate the outliers and those with abnormal TSH and CRP values. Hence, total of 252 subjects were included in the study. After exclusion of 17 subjects on basis of serum TSH and CRP values, total subjects analysed were finally 235.
Procedure
All the included study participants were evaluated for their health status by history and clinical examination. A proforma was used for this purpose. A 4 mL of venous blood was drawn by aseptic precautions and serum was separated within 3 hours of blood collection. The estimation of serum Cystatin C and sCr along with other tests to establish exclusion criteria i.e., TSH and CRP was completed within 12 hours of blood collection.
• Serum Cystatin C was evaluated by latex enhanced immunoturbidimetric cystatin C assay using a calibrator traceable to international standard ERM-DA471 developed by the International Federation of Clinical Chemistry (IFCC) and Laboratory Medicine (21).
• Serum creatinine by Jaffe method in a fully automatic clinical chemistry analyser.
• TSH by electro chemiluminometer (Roch).
• CRP by immunoturbidimetry.
Cystatin C values of euthyroid and CRP negative (<10 mg%) subjects were used to determine the reference interval.
Statistical Analysis
MedCalc free trial version 18.9 was used for data analysis. There were no outliers identified by Turkey’s method using MedCalc statistical software. Categorical variables are presented using frequency (percentage) while continuous variables are summarised with an interpercentile range. The Kolmogorov-Smirnov test was used to assess the normality of the distribution of continuous variables. Reference Intervals (RIs) were determined by the non parametric method as described in the IFCC and Clinical and Laboratory Standard Institute (CLSI) guidelines (20),(21). This method was used to determine the 2.5 and 97.5 percentiles and the respective 90% Confidence Intervals (CI) around these estimates.
Results
The data obtained from 235 subjects were analysed. The age of the study subjects ranged from 20 to 60 years and the majority fall in the age group of 20 to 25 years. The body weight of the study subjects were in the range of 39 to 125 kg. Out of 235 subjects, 17 were males and 218 were females (Table/Fig 1).
The non parametric reference intervals for serum Cystatin C are found to be from 0.39 to 0.79 mg% and that of sCr ranged from 0.79 to 1.2 mg%. The 90% confidence intervals for the upper and lower reference limits are shown in (Table/Fig 2).
The frequency distribution of serum Cystatin C levels for the reference individuals is shown in (Table/Fig 2). The relationship between Cystatin C with age is displayed in (Table/Fig 3), which shows an increase in Cystatin C levels as age advances; Spearman’s rank correlation coefficient (rho=0.197). Similarly, the weak correlation between Cystatin C and sCr was observed (rho=0.3701) (Table/Fig 4).
Discussion
Cystatin C refers to an alkaline, non glycosylated protein,13 KD (Kilo Dalton), protease inhibitor, that is expressed continuously in the nuclei of all cells (5). They can cross the glomerular filter membrane in the physiological environment, and is completely reabsorbed and degraded in the renal tubules without recirculation and the renal tubules does not secrete the protein-Cystatin (11),(14). It has been seen that under physiological conditions, there is very low concentration of cystatin C in urine and blood. However, this concentration increases when the renal tubules are damaged and are dysfunctional (22),(23).
Cystatin C has proven to be a good indicator of renal diseases with help of rapid, sensitive and precise immunoassays, it can be used for diagnostic purposes in such cases (24). In this study, reference intervals for plasma Cystatin C in adults were evaluated using latexenhanced immunoturbidimetry based standardised Cystatin C assay enabling traceability to the international standard ERM-DA471 (21). The reference interval of Cystatin C among the reference individuals enrolled in this study was found to be 0.39 to 0.79 mg%.
In a similar study by Norlund L et al., the authors observed that, the mean value was 1.08 mg% in male patients and 1.03 mg% for female patients. They showed a gender difference (0.05 mg%); not statistically significant, owing to the difference in population (25). In the present study, no separate reference interval for males and females could be found due to an inadequate sample size for male patients.
Another analogous study conducted by Finney H et al., 309 participants exhibited a noteworthy difference between values for males (mean 0.74 mg%) and females (mean 0.68 mg%), although the range for the distributions was identical (0.42 mg%) and hence discrete ranges were not required (24). The reference interval obtained for Cystatin C latex-enhanced needs immunoturbidimetry in the present study was found to be 0.39-0.79 mg%, which was almost similar to the outcome of Finney H et al., with a value of 0.51-0.98 mg/L; done by latex particle enhanced immune nephelometric assay (latex PENIA) (24). Another similar study by Groesbeck D et al., also showed that the mean serum Cystatin C level was 0.84 mg% and was higher among the males than the females (26).
On the contrary, a study that was done by Pergande M and Jung K, reported significantly lower levels of Cystatin C (1.78±0.26) in females than that in males (2.14±0.31) but since their sample size was small with 33 subjects in both females and males, their study results did not match the present study outcome (27). The Cystatin C range in the present study was found to be different from other studies conducted by Andersen KJ et al., with a value of 0.61-1.21 mg% both in healthy and diseased conditions, Erlandsen EJ et al., with a value of 0.54-1.21mg/L in both male and female population, and Edinga-Melenge BE et al., with a value of 0.60-1.10 mg/L, wherein the particle-enhanced turbidimetric immune assay was used in camaroonian adult population (11),(28),(29).
Few other studies that recorded the reference range of Cystatin C were done by Kottgen A et al., with a value of 0.61-1.04 mg% done in United States population (30), Li DD et al., value of 0.60-1.08 mg% in a Chinese population (31) and Okonkwo IN et al., in a Nigerian population with a value of 0.64-1.12 mg% (32). The present study consisted of subjects belonging to the Dravidian population subset of South Asian ethnic groups revealed a reference interval of 0.39-0.79 mg/L which was found to be lower than the other ethnic groups (30),(31),(32),(33).
The study conducted by Edinga-Melenge BE et al., explained that the serum Cystatin C levels had a tendency to be slightly affected by certain distinguishing factors like age and gender (29). However, the influence of gender on the various range levels of this specific protein still remains unclear. However, there are certain studies that have reported that the levels of Cystatin C were independent of gender variances while few other have stated their direct significant influence on the same (33),(34),(35),(36). Edinga-Melenge BE et al., found that gender formed one autonomous illustrative feature for the values of serum Cystatin C (29). The present study showed that serum cystatin C levels were 11% higher in males than in females (0.90 mg/L vs 0.80 mg%; p-value <0.001). Similar results were also observed by Kottgen A et al., who showed a difference of 8% between males and females (30). However, Al Wakeel JS et al., reported lower levels of serum cystatin C in males as compared to females (0.72 mg% vs 0.77 mg%; p-value <0.001) in a Saudi population (27). Hence, it can be deduced that a single adult reference range for Cystatin C, with distinct clinically significant differences between the males and females was obtained (37). Thus, through multicentre study of the baseline levels of serum cystatin C, it creates the diagnostic concentration reference interval of the protein in order to encourage the recognition, standardisation and establishment of specific serum Cystatin C levels.
Limitation(s)
Due to the non random sampling method used and single measurement of serum cystatin C levels, generalisation of the present findings to the entire Dravidian south Asian population cannot be done as the representatives of the study population would have been better obtained with randomisation. Secondly, a separate reference interval for males and females could not be found due to unequal division of study participants amongst both the genders.
Conclusion
This study depicted serum Cystatin C in Dravidian population subset of South Asian ethnic population and showed that males had suggestively higher levels of Cystatin C as compared to the females. The reference limits of serum Cystatin C in the study population belonging to the South Asian ethnic groups was observed as 0.39-0.79 mg%. Hence, it is expected and assumed that the present study data would further instigate other researchers to conduct similar studies on a larger population that would represent the population of a whole country of nation with vivid diversity.
Reference
| 1. | Collins AJ, Foley RN, Herzog C, Chavers B, Gilbertson D, Ishani A, et al. US Renal Data System 2010 Annual Data Report. Am J Kidney Dis. 2011;57(1 Suppl 1):A8-e1-526.
[ CrossRef] [ PubMed] | 2. | Singh AK, Farag YM, Mittal BV, Subramanian KK, Reddy SR, Acharya VN, et al. Epidemiology and risk factors of chronic kidney disease in India - results from the SEEK (Screening and Early Evaluation of Kidney Disease) study. BMC Nephrol. 2013;14:114. Doi: 10.1186/1471-2369-14-114.
[ CrossRef] [ PubMed] | 3. | National Kidney Foundation. K/DOQI clinical practice guidelines for chronic kidney disease: evaluation, classification, and stratification. Am J Kidney Dis. 2002;39(2 Suppl 1):S1-266.
| 4. | European Best Practice Guidelines Expert Group on Hemodialysis, European Renal Association. Section I. Measurement of renal function, when to refer and when to start dialysis. Nephrol Dial Transplant. 2002;17(Suppl 7):07-15.
| 5. | Cockcroft DW, Gault MH. Prediction of creatinine clearance from serum creatinine. Nephron. 1976;16(1):31-41.
[ CrossRef] [ PubMed] | 6. | Shemesh O, Golbetz H, Kriss JP, Myers BD. Limitations of creatinine as a filtration marker in glomerulopathic patients. Kidney Int. 1985;28(5):830-38.
[ CrossRef] [ PubMed] | 7. | Grubb A, Simonsen O, Sturfelt G, Truedsson L, Thysell H. Serum concentration of cystatin C, factor D and beta 2-microglobulin as a measure of glomerular filtration rate. Acta Med Scand. 1985;218(5):499-503.
[ CrossRef] [ PubMed] | 8. | Grubb A, Löfberg H. Human gamma-trace, a basic microprotein: Amino acid sequence and presence in the adenohypophysis. Proc Natl Acad Sci U S A. 1982;79(9):3024-27.
[ CrossRef] [ PubMed] | 9. | Barrett AJ, Fritz H, Grubb A, Isemura S, Järvinen M, Katunuma N, Machleidt W, Müller-Esterl W, Sasaki M, Turk V. Nomenclature and classification of the proteins homologous with the cysteine-proteinase inhibitor chicken cystatin. Biochem J. 1986;236(1):312.
[ CrossRef] [ PubMed] | 10. | Simonsen O, Grubb A, Thysell H. The blood serum concentration of cystatin C (gamma-trace) as a measure of the glomerular filtration rate. Scand J Clin Lab Invest. 1985;45(2):97-101.
[ CrossRef] [ PubMed] | 11. | Andersen KJ, Schmidt C, Nordin G, Andersson B, Nilsson-Ehle P, Lindström V, et al. Serum cystatin C, determined by a rapid, automated particle-enhanced turbidimetric method, is a better marker than serum creatinine for glomerular filtration rate. Clin Chem. 1994;40(10):1921-26.
[ CrossRef] | 12. | Newman DJ, Thakkar H, Edwards RG, Wilkie M, White T, Grubb AO, et al. Serum cystatin C measured by automated immunoassay: A more sensitive marker of changes in GFR than serum creatinine. Kidney Int. 1995 ;47(1):312-18.
[ CrossRef] [ PubMed] | 13. | Finney H, Newman DJ, Gruber W, Merle P, Price CP. Initial evaluation of cystatin C measurement by particle-enhanced immunonephelometry on the Behring nephelometer systems (BNA, BN II). Clin Chem. 1997;43(6 Pt 1):1016-22.
[ CrossRef] [ PubMed] | 14. | Curhan G. Cystatin C: A marker of renal function or something more? Clin Chem. 2005;51(2):293-94.
[ CrossRef] [ PubMed] | 15. | Shukla AN, Juneja M, Patel H, Shah KH, Konat A, Thakkar BM, et al. Diagnostic accuracy of serum cystatin C for early recognition of contrast induced nephropathy in Western Indians undergoing cardiac catheterization. Indian Heart J. 2017;69(3):311-15.
[ CrossRef] [ PubMed] | 16. | Demirtas¸ S, Bozbas¸ A, Akbay A, Yavuz Y, Karaca L. Diagnostic value of serum cystatin C for evaluation of hepatorenal syndrome. Clin Chim Acta. 2001;25;311(2):81-89.
[ CrossRef] [ PubMed] | 17. | O’Riordan SE, Webb MC, Stowe HJ, Simpson DE, Kandarpa M, Coakley AJ, et al. Cystatin C improves the detection of mild renal dysfunction in older patients. Annals of Clinical Biochemistry. 2003;40(6):648-55.
[ CrossRef] [ PubMed] | 18. | Jernberg T, Lindahl B, James S, Larsson A, Hansson LO, Wallentin L, Cystatin C. A novel predictor of outcome in suspected or confirmed non-ST-elevation acute coronary syndrome. Circulation. 2004;110(16):2342-48.
[ CrossRef] [ PubMed] | 19. | Shlipak MG, Katz R, Fried LF, Jenny NS, Stehman-Breen C, Newman AB, et al. Cystatin-C and mortality in elderly persons with heart failure. J Am Coll Cardiol. 2005;45(2):268-71.
[ CrossRef] [ PubMed] | 20. | Knight EL, Verhave JC, Spiegelman D, Hillege HL, de Zeeuw D, Curhan GC, et al. Factors influencing serum cystatin C levels other than renal function and the impact on renal function measurement. Kidney Int. 2004;65(4):1416-21.
[ CrossRef] [ PubMed] | 21. | Henny J. The IFCC recommendations for determining reference intervals: Strengths and limitations / Die IFCC-Empfehlungen für die Bestimmung von Referenzbereichen: Stärken und Schwächen. Laboratoriums Medizin. 2009;33(2):45-51.
[ CrossRef] | 22. | Zhou W, Zhou LL. The clinical value of urine cystatin C combined with serum β_2 microglobulin level in the diagnosis of patients with lupus nephritis. Labeled Immunoassay and Clinic. 2012;2(1):169-76.
[ CrossRef] [ PubMed] | 23. | Jiang X, Qin L, Su Guosheng, Su X, Lu Aihua, Huang J, Lu Feng, Lu T, Huamg P, Sub UQING. Urine Cystatin C determination in the establishment of reference interval in the diagnosis and treatment of Renal injury. Natural Science. 2022;14(1):13-17.
[ CrossRef] | 24. | Finney H, Newman DJ, Price CP. Adult reference ranges for serum cystatin C, creatinine and predicted creatinine clearance. Ann Clin Biochem. 2000;37(Pt 1):49-59.
[ CrossRef] [ PubMed] | 25. | Norlund L, Fex G, Lanke J, Von Schenck H, Nilsson JE, Leksell H, Grubb A. Reference intervals for the glomerular filtration rate and cell-proliferation markers: Serum cystatin C and serum beta 2-microglobulin/cystatin C-ratio. Scand J Clin Lab Invest. 1997;57(6):463-70.
[ CrossRef] [ PubMed] | 26. | Groesbeck D, Köttgen A, Parekh R, Selvin E, Schwartz GJ, Coresh J, Furth S. Age, gender, and race effects on cystatin C levels in US adolescents. Clin J Am Soc Nephrol. 2008;3(6):1777-85.
[ CrossRef] [ PubMed] | 27. | Pergande M, Jung K. Sandwich enzyme immunoassay of cystatin C in serum with commercially available antibodies. Clin Chem. 1993;39(9):1885-90.
[ CrossRef] [ PubMed] | 28. | Erlandsen EJ, Randers E, Kristensen JH. Reference intervals for serum cystatin C and serum creatinine in adults. Clin Chem Lab Med. 1998;36(6):393-97.
[ CrossRef] [ PubMed] | 29. | Edinga-Melenge BE, Yakam AT, Nansseu JR, Bilong C, Belinga S, Minkala E, et al. Reference intervals for serum cystatin C and serum creatinine in an adult sub- Saharan African population. BMC Clin Pathol. 2019;19:4. Doi: 10.1186/ s12907- 019-0086-7.
[ CrossRef] [ PubMed] | 30. | Köttgen A, Selvin E, Stevens LA, Levey AS, Van Lente F, Coresh J. Serum cystatin C in the United States: The Third National Health and Nutrition Examination Survey (NHANES III). Am J Kidney Dis. 2008;51(3):385-94.
[ CrossRef] [ PubMed] | 31. | Li DD, Zou MN, Hu X, Zhang M, Jia CY, Tao CM, Wang LL, Ying BW. Reference intervals and factors contributing to serum cystatin C levels in a Chinese population. J Clin Lab Anal. 2012;26(2):49-54.
[ CrossRef] [ PubMed] | 32. | Okonkwo IN, Ogbu II, Ijoma UN, Ulasi II, Ijoma CK. Reference intervals for serum cystatin C and creatinine of an indigenous adult Nigerian population. Niger J Clin Pract. 2015;18(2):173-77.
[ CrossRef] [ PubMed] | 33. | Ijoma C, Ijoma U, Okonkwo I, Ogbu I, Ulasi I. Reference intervals for serum cystatin C and creatinine of an indigenous adult Nigerian population. Niger J Clin Pract. 2015;18(2):173.
[ CrossRef] [ PubMed] | 34. | Delanaye P, Chapelle J-P, Gielen J, Krzesinski J-M, Rorive GL. intérêt de la cystatine C dans l’évaluation de la fonction rénale. Néphrologie. 2003;24:457-68.
| 35. | Wasén E, Suominen P, Isoaho R, Mattila K, Virtanen A, Kivelä SL, Irjala K. Serum cystatin C as a marker of kidney dysfunction in an elderly population. Clin Chem. 2002;48(7):1138-40.
[ CrossRef] [ PubMed] | 36. | Maahs DM, Prentice N,McFann K,Snell-bergeon JK, Jalal D, Bishop FK, et al. Age and Sex Influence Cystatin C in adolescents with and without type 1 diabetes. Diabetes Care. 2011;34(11):2360-62.
[ CrossRef] [ PubMed] | 37. | Al-Wakeel JS, Memon NA, Chaudhary AR, Mitwalli AH, Tarif N, Isnani A, Hammad D. Normal reference levels of Serum Cystatin C in Saudi Adults. Saudi J Kidney Transpl. 2008;19(3):361-70. |
DOI: 10.7860/JCDR/2022/58894.16965
Date of Submission: Aug 09, 2022
Date of Peer Review: Sep 23, 2022
Date of Acceptance: Sep 30, 2022
Date of Publishing: Oct 01, 2022
AUTHOR DECLARATION:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? Yes
• For any images presented appropriate consent has been obtained from the subjects. NA
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