|Year : 2017 | Volume
| Issue : 6 | Page : 1247-1255
|Serum cystatin C levels in Healthy Nigerian neonates: Is there a need for normative values in Nigerian babies?
Akpoembele D Madise-Wobo1, Olusegun H Gbelee2, Adaobi Solarin1, Barakat A Animasahun2, Olisamedua F Njokanma2
1 Department of Pediatrics, Lagos State University Teaching Hospital, Ikeja Lagos, Nigeria
2 Department of Pediatrics, Lagos State University Teaching Hospital; Department of Pediatrics and Child Health, Lagos State University College of Medicine, Ikeja Lagos, Nigeria
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|Date of Web Publication||18-Dec-2017|
| Abstract|| |
Cystatin C is an endogenous marker of renal function. Normal reference values have been documented in neonates outside Africa, but no study has been documented in African neonates. With reports that race may affect serum cystatin C values, this study was carried out to generate normal values in apparently healthy term neonates at birth and three days of life neonates in Nigeria. This was a hospital-based prospective study. A cohort of 120 apparently healthy term neonates were recruited at birth. Serum cystatin C was measured from the cord blood at birth and venous blood when they were three days old using enzyme-linked immunosorbent assay (ELISA) method. The mean serum cystatin C values for cord blood and 3rd day venous samples were 1.67 ± 0.52 mg/L and 1.62 ± 0.52 mg/L, respectively (P = 0.87). The cord blood and 3rd day serum cystatin C values for males were 1.67 ± 0.47 mg/L and 1.68 ± 0.51 mg/L, respectively (P = 0.77) and the values for females were 1.68 ± 0.56 mg/L and 1.58 ± 0.52 mg/L, respectively (P = 07.22). The serum cystatin C levels were similar among the different birth weight groups and gestational age (P >0.05). The cord blood and 3rd day serum cystatin C values were similar. Serum cystatin C values were independent of gender and birth weight of neonates. The values of serum cystatin C in Nigerian neonates were comparable to that reported for neonates in other regions of the world. It is recommended that ELISA technique may be reliably used to measure serum cystatin C levels in neonates.
|How to cite this article:|
Madise-Wobo AD, Gbelee OH, Solarin A, Animasahun BA, Njokanma OF. Serum cystatin C levels in Healthy Nigerian neonates: Is there a need for normative values in Nigerian babies?. Saudi J Kidney Dis Transpl 2017;28:1247-55
|How to cite this URL:|
Madise-Wobo AD, Gbelee OH, Solarin A, Animasahun BA, Njokanma OF. Serum cystatin C levels in Healthy Nigerian neonates: Is there a need for normative values in Nigerian babies?. Saudi J Kidney Dis Transpl [serial online] 2017 [cited 2020 Jul 9];28:1247-55. Available from: http://www.sjkdt.org/text.asp?2017/28/6/1247/220881
| Introduction|| |
The measurement of glomerular filtration rate (GFR) is an important part of the clinical evaluation of renal function.,,, An assessment of renal function may be required for several reasons: to identify renal impairment, monitor disease progression, and assess baseline measurements prior to starting treatment with certain drugs and adjustment of medications based on GFR changes.
The gold standard method for measuring GFR is to measure the clearance of inulin and other exogenous markers which are freely filtered by the glomerulus and are not metabolized, reabsorbed, or secreted by the renal tubular cells., However, frequent blood sampling, urine collection, and constant infusion of exogenous markers limit their use. In addition, these methods are time consuming, expensive, and cumbersome.
GFR is also evaluated indirectly by the clearance of the endogenous substance creatinine. The source of creatinine is creatine and phosphocreatine of skeletal muscles. Thus, the level of serum creatinine is a reflection of the muscle mass of the individual. Although serum creatinine is widely used to estimate GFR, it is not without many drawbacks. Firstly, growth and changes in muscle mass influence serum creatinine levels.,, Secondly, serum creatinine at birth reflects maternal serum creatinine because it crosses the placenta., Thirdly, because of the immature renal tubules at birth, some creatinine is reabsorbed by the tubules resulting in a higher serum creatinine in the immediate neonatal period, leading to an underestimation of the true GFR in neonates. Fourthly, the most commonly available method used to measure serum creatinine, Jaffe method, has interference from conditions such as hyperbilirubinemia, hemolysis, and ketone bodies in the blood., This leads to a falsely high serum creatinine and an underestimation of GFR. Finally, serum creatinine is not sensitive to small changes in GFR. These reasons constitute disadvantages to the use of creatinine to estimate GFR in the newborn.
Cystatin C has emerged as a result of a search for a more precise endogenous marker of GFR., Cystatin C is a 122 amino acid, 13 Kilo Dalton protein, that is a member of the family of cysteine protease inhibitor.,, It is a product of the housekeeping gene expressed in all nucleated cells and produced at a constant rate in the body. It is freely filtered by the glomerulus and catabolized in the proximal tubules.,,, Unlike creatinine, it does not cross the placenta and is not affected by the serum level of bilirubin, hemolysis, or ketones. It is independent of muscle mass, age, gender, and nutritional status., Its level in the blood is thus a more precise reflection of the function of the kidneys.,
Cystatin C values can be measured in a random sample of serum or plasma using immunoassays such as immunonephelometry, immunoturbidimetry, and enzyme-linked immunosorbent assay (ELISA). Most studies with documented reference values in neonates have utilized immunonephelometry and immunoturbidimetry assay techniques. Those commonly used assay techniques are not readily available in most developing countries in sub-Saharan Africa. Although the use of ELISA technique is not as popular as the other assay techniques, it has some advantages over the other two methods. ELISA technique is easy to perform and measures very low concentration of cystatin C compared to both immunonephelometry and immunoturbidimetry. Furthermore, ELISA is affordable and readily available in resource-poor countries.
A number of studies have documented the reference values of serum cystatin C in term and preterm neonates outside Africa with varying reports. While some authors investigated serum cystatin C values at birth and 3rd day of life,,,, others have reported values from birth to the end of the 1st month of age.,, The general trend noted was that cystatin C values were higher at birth, and those values were higher than the documented normal values for older infants, children, and adults.,,, The values of serum cystatin then declined gradually until the end of the 1st year, when it becomes comparable with adult values.,, It has been suggested that the high normal serum level of cystatin C at birth, usually higher than the maternal level,, mirrors the maturation of the kidneys. Furthermore, while some researchers have reported that there was no significant difference in the serum cystatin C values at birth and 3rd day,,, or in the 1st week, or up to end of the 1st month of life,, others have noted that there was a significant difference in the values at birth and 3rd day of life.
Standard values of serum cystatin C have been documented in neonates in South America and Europe, but no study has yet been documented in Africa. Furthermore, literature search has shown variations in the values of cystatin C as highlighted in the previous paragraph. There are also reports that race might affect serum cystatin C levels.,, It is, therefore, important to study the values of serum cystatin C in African neonates utilizing the readily available ELISA technique. These can then be used as reference values when assaying cystatin C as a marker of renal impairment in the newborn.
| Subjects and Methods|| |
Study design and setting
This was a prospective study of apparently healthy term neonates. The study was carried out in the delivery suites, labor ward theater, and the postnatal ward of the General Hospital, Ifako Ijaiye. The study site is a secondary health facility, an annex of the Lagos State University Teaching Hospital (LASUTH), with an average number of deliveries of 300/month. LASUTH is a tertiary hospital in south-western Nigeria.
Clearance was obtained from the Health Research and Ethics Committee of LASUTH. Signed written informed consent was obtained from the parents of the neonates.
Babies were included according to the following criteria: apparently healthy term neonates delivered at the study site (these were neonates without fever or any other signs and symptoms of illness), babies with 5 min Apgar scores of more than seven, and babies of mothers who gave written informed consent for participating in the study.
The following babies were excluded: those babies born to mothers with renal failure, chronic or pregnancy-induced hypertension, babies with congenital anomalies,, babies of mothers with a history of polyhydramnios or oligohydramnios during antenatal period, those babies who presented with abdominal distension and ballotable kidneys, and babies who were apparently healthy at the time of delivery but developed fever or signs and symptoms of systemic illness by three days of postnatal age.
Sample size was calculated using the following formula for estimation of a single mean as follows:
where n = sample size
Z = Standard deviation (SD) at 95% confidence interval (1.96)
α2 = Population variance.
t2 = Level of precision.
From a previous study, the mean ± SD of serum cystatin C for term babies on the 3rd day of life was 1.70 ± 0.26 mg/L. Assuming a 5% degree of precision of the mean and confidence coefficient of 95%,
Therefore n = 103
To accommodate possible attrition from unforeseen circumstances, an additional 10% of the total sample size, 10 was added. Therefore, the final sample size was 113 which was rounded up to 120.
Relevant antenatal information was recorded from the mothers of the babies using a self-designed proforma. At birth, cord blood sample was obtained following the guidelines for obtaining cord blood samples by the American Congress of Obstetricians and Gynaecologists. Gestational age was determined from the 1st day of the mother's last regular menstrual period (LMP) and corroborated by Ballard score., When discrepancy between the gestational age from LMP and Ballard score was more than two weeks, the gestational age estimated by the Ballard score was used. The babies were examined and had Apgar score at 1 and 5 min documented and then weighed. At three days of age, babies who were recruited for the study were re-examined. Those who were apparently healthy had venipuncture performed according to the WHO guidelines.
In batches of 80 samples, serum cystatin C was measured on the cord and 3rd day blood samples using human cystatin C ELISA kits manufactured by BioVendor-Laboratorni a.s, from the Czech Republic. The calibration range of the assay was 200 to 10,000 ng/mL with a limit of detection of 0.25 ng/mL. Its intra-assay coefficient of variation was 2.5% while the inter-assay coefficient of variation was 6.9%.
Test of normality was performed by means of the Kolmogorov–Smirnov and Shapiro–Wilk tests. Measures of statistical location such as the mean and SD were calculated for variables that were normally distributed. Variables with skewed distribution were summarized with median. Comparisons between groups was made by the paired Student t-test, independent t-test, and ANOVA for normally distributed data while the Wilcoxon signed-rank, Mann–Whitney U-test, and Kruskal–Wallis tests were used for nonparametric data.
| Results|| |
Demographic characteristics of the study subjects
A total of 120 babies had cord blood and 3rd day samples collected, but results were available for 113 babies because seven babies whose samples hemolyzed, were excluded. Ninety-eight babies (86.7%) were products of booked pregnancies. By the study design, all the babies were term deliveries. [Table 1] illustrates the demographic characteristics of the study subjects. There were a total of 45 males and 68 females, with a male-to-female ratio of 1:1.5. The modal gestational age at delivery for both sexes was 38 weeks, with a mean of 38.8 ± 1.3 weeks. The babies weighed 2.5 kg to 4.5 kg at birth, with a mean weight of 3.4 ± 0.5 kg. There was no significant difference in the birth weights between the two sexes (P = 0.88).
Cystatin C levels
The serum cystatin C values (mean ± SD), at birth and three days of life, were 1.67 ± 0.52 and 1.62 ± 0.52 mg/L with medians of 1.8 and 1.6, respectively. There was no significant difference between the values at birth and three days of life (P = 0.39). [Table 2] shows the cord blood and three-day cystatin C values for males and females. The values of cystatin C for the males and females were similar. Among both males and females, there was no significant difference in the cord blood and 3rd day cystatin C values (P >0.05).
|Table 2: Serum cystatin C values at birth and three days of age, by gender.|
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The range of values for cystatin C for both cord blood and day three samples was 0.60–2.60 mg/L and 0.43–2.60 mg/L, respectively. However, for ease of description, the cord blood and day three cystatin C values were grouped into various sub-ranges as shown in [Table 3]. The proportions of cystatin C were similar across the different subgroups (P = 0.402). However, the cord blood samples had more values in the subgroup of ≥2 mg/L compared to the 3rd day samples.
|Table 3: Comparison of serum cystatin C sub-range values with respect to cord blood and 3rdday venous sample.|
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Concerning the distribution of the values of serum cystatin C in relation to gestational age, there was a gradual increase in the cystatin C values at birth as the gestational age increased, but the difference was not significant (P >0.05). A similar pattern was noted for the 3rd day samples. This is shown in [Table 4].
|Table 4: Serum cystatin C values at birth and 3rd day of life based on gestational age.|
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[Table 5] shows the birth weight groups and serum cystatin C levels at birth and three days of life. There was no significant difference between the mean cystatin C values at birth and at three days of life among all birth weight subgroups (P >0.05).
|Table 5: Serum cystatin C at birth and three days of age based on birth weight.|
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Considering that there was no gender difference or difference between the cord and 3rd day samples, a single mean value for both cord blood and 3rd day venous sample of serum cystatin C was calculated. The mean and SD for cystatin C was 1.65 ± 0.52 with a median of 1.7 mg/L.
| Discussion|| |
This is the first study to document serum cystatin C values and assess kidney function in term newborn babies at birth and three days postnatal age, using serum cystatin C in Nigeria. Careful literature search did not reveal any previously published reports on cystatin C in newborn in Nigeria. The current study, therefore, provides the much-needed information, especially as cystatin C has proven advantages over creatinine-based methods.,,,,,
The mean value of serum cystatin C at birth and three days of life documented in this study is consistent with other reports., The values were in the highest range of normal for children without renal abnormalities. It is known that serum cystatin C values are highest at birth and then decline gradually until the end of the 1st year when they are comparable with adult levels.,, It was, therefore, not surprising that the values obtained in the present study were higher than that reported for older children and adults.
The mean serum cystatin C levels herein reported are comparable with some,, higher than some others,, and lower than other reported values., The present study and the research by Bahar et al and Treiber et al had comparable subjects with subtle differences in the methods. The difference in the mean values between the present study and that by Bahar et al and Treiber et al may be explained by the difference in assay techniques among the studies. ELISA was used in the present study while immunonephelometry was used in the earlier studies. It has been suggested that difference in assay techniques may affect the values of serum cystatin C.,,, However, the studies with comparable mean values with the present study also used different assay techniques compared with the present study. For example, Novo et al in Brazil used the same assay technique as Bahar et al which was immunonephelometry, but their values were not significantly different from the values of cystatin C in the present study. Similarly, Harmoinen et al in Germany used immunoturbidimetry, an assay technique that was different from ELISA used in the present study and immunonephelometry used by Novo et al; however, all the three studies had comparable results. Thus, difference in assay technique alone may not explain the difference in the serum cystatin C values among various studies. Another possible explanation for the difference in results lies in the difference in calibration with the different assay techniques used to assay cystatin C. It has been shown that difference in values assigned to different calibrator materials used to assay cystatin C also contributes to inter-laboratory difference in the results of measured cystatin C [Table 6].,,,
|Table 6: Literature extraction of reference values of serum cystatin C in neonates.|
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The difference in the mean values of cystatin C in the present study compared to the report by Finney et al may be explained, in addition to the previously documented reasons, by the difference in the characteristics of study subjects in both studies. The subjects in the earlier study were 0–3 months of age compared to 0–3-day-old neonates in the present study. Cystatin C values are known to be highest in the first few days of life with a gradual decline during the first four months of life. The lower values in the earlier report may have been contributed by the older infants in that study.
The levels of serum cystatin C in the present study were lower than an earlier report by Treiber et al in Slovenia. A possible explanation lies in the difference in the characteristics of the study subjects between the two studies. While the present study subjects were term babies, the earlier researchers investigated preterm and term newborn babies. It has been shown that preterm babies have higher values of serum cystatin C than term infants.,, It is thus possible that the higher values of cystatin C in the earlier research by Treiber et al may have been contributed by the preterm neonates.
Given the difference in the mean values of serum cystatin C, one area where one might seek explanation for the differences in the levels of serum cystatin C in the neonates may be the difference in the races of the study subjects.
Comparing the values of serum cystatin C from Europe with results from South America revealed varying results. However, even within the same geographic regions and study populations in Europe, that is, the UK, Slovenia, Germany, and Denmark, there were variations in the values of serum cystatin C. Second, differences in the selection of subjects and methods also contributed to the varying differences as explained earlier.
The cord blood at birth and 3rd day serum cystatin C values in the present study were comparable. The serum cystatin C values dropped slightly from birth to the three-days-old samples, but the difference was not significant. This finding is consistent with results from previous studies.,,, Cord blood and 3rd day serum cystatin C values were comparable because, although the cystatin C values declined gradually in the 1st year, there are no significant changes in its values in the first few days of life. Furthermore, there is no significant change in the GFR within the first three days of life. Thus, it was not surprising that the cord blood and 3rd day serum cystatin C values were not significantly different. The implication of this finding is that there will be no need to measure different values of serum cystatin C at birth and the 3rd day of life.
There were other findings of interest noted in the present study. Firstly, the documented range of serum cystatin C in the present study was wide, from <1 mg/L to more than 2 mg/L. However, more than 90% of the values were more than 1 mg/L. A similar finding of widely dispersed cystatin C values in neonates has been documented in previous studies.,
Secondly, concerning the mean values of cystatin C in relation to the birth weight and gender of the babies, there was no significant difference in the values of cystatin C when compared as a total group and subgroups. This finding is in agreement with previous reports that serum cystatin C is independent of weight and gender.,,, This is one of the advantages of cystatin C over creatinine, being independent of gender and weight. Thus, irrespective of the birth weight and gender of the neonate, there is no significant difference in the cystatin C values in the first few days of life, and a separate reference value need not be generated for different sex and weight of babies.
Lastly, gestational age of the term babies did not influence the serum cystatin C values in the present study. Other researchers have documented insignificant difference in the values of cystatin C across preterm and term infants., Considering that there was no significant difference in mean cystatin C among the gestational age groups of the term babies in this study, there will be no need to generate reference values for different gestational ages of term neonates.
| Conclusion|| |
This study has generated normal values of cystatin C for apparently healthy Nigerian babies at birth and at 3rd day of life using ELISA technique. The values were comparable with previously reported values, obtained from more sophisticated techniques such as nephelometry and turbidimetry, higher than some and lower than others. There was no significant difference between the cord blood and 3rd day levels of serum cystatin C. Similarly, the cystatin C values were unaffected by the gender, gestational age, and birth weights of the babies.
Conflict of interest: None declared.
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Akpoembele D Madise-Wobo
Department of Pediatrics, Lagos State University Teaching Hospital, Ikeja Lagos
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]
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