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 Table of Contents  
Year : 2021  |  Volume : 28  |  Issue : 1  |  Page : 39-43

Ionised serum calcium reference interval among rural women of reproductive age in Abuja, Nigeria

1 Department of Obstetrics and Gynaecology, University of Abuja Teaching Hospital, Abuja, Nigeria
2 Department of Community and Public Health, University of Abuja Teaching Hospital, Abuja, Nigeria
3 Department of Chemical Pathology, University of Abuja Teaching Hospital, Abuja, Nigeria

Date of Submission23-Aug-2020
Date of Decision27-Dec-2020
Date of Acceptance26-Jan-2021
Date of Web Publication25-Feb-2021

Correspondence Address:
Dr. Habiba Ibrahim Abdullahi
Department of Obstetrics and Gynaecology, University of Abuja Teaching Hospital, Abuja
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/npmj.npmj_274_20

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Background: Various medical conditions in pregnancy may be traced to suboptimal levels of serum calcium during pregnancy. Communities' derivation of normal serum calcium reference interval is imperative. Objective: The objective was to determine the normal free (ionised) serum calcium reference interval among women of reproductive age in the federal capital territory (FCT) of Nigeria. Materials and Methods: This was a cross-sectional study of 240 women from six primary health-care facilities in Abuja, Nigeria. Their blood samples were collected in serum separator bottles. The outcome measure was the determination of the reference interval of free (ionised) serum calcium among the participants. Results: The obtained normal reference interval of ionised serum calcium (iCa) was 0.88–1.4 mmol/L. The accompanying reference intervals for total protein and albumin were 5.7–9.4 mg/dl and 3.3–5.2 mg/dl, respectively. Conclusion: The derived reference interval of iCa in this study was 0.88–1.4 mmol/L, while the total calcium range was 2.18–2.82 mmol/L. These intervals are recommended for use in the Nigerian FCT and its environs as it provides lower intervals compared to the operational values. There is a need for a national derived value as this may change the practice.

Keywords: Federal capital territory, reproductive age, serum calcium, women

How to cite this article:
Onyekwelu AC, Abdullahi HI, Isah AY, Jamda AM, Nwegbu MM. Ionised serum calcium reference interval among rural women of reproductive age in Abuja, Nigeria. Niger Postgrad Med J 2021;28:39-43

How to cite this URL:
Onyekwelu AC, Abdullahi HI, Isah AY, Jamda AM, Nwegbu MM. Ionised serum calcium reference interval among rural women of reproductive age in Abuja, Nigeria. Niger Postgrad Med J [serial online] 2021 [cited 2022 Jan 27];28:39-43. Available from: https://www.npmj.org/text.asp?2021/28/1/39/310157

  Introduction Top

Calcium is essential for bone mineralisation, neuromuscular function and secretion of hormones and enzymes that enables normal functions of virtually all organs of the body.[1] Skeleton and teeth are traditionally a reservoir of over 98% of total calcium in the body and hence, readily available sources of serum calcium.[1] About 50% of the blood calcium is ionised, while the rest is protein bound. The normal reference value for total calcium in the blood is 9–11 mg/100 ml (4.5–5.3 mEq/L).[2] It has, however, been established that reference value may vary with age, sex, race, pregnancy, diet, use of prescribed or herbal drugs and stress.[3] Therefore, determination of serum calcium reference interval among women of reproductive age in a particular subregion such as Nigerian's capital community appears pertinent, more so that previous similar research standard on the subject matter appears unavailable for our environment to the best of the researcher's knowledge. A reference range is usually defined as the set of values that 95 percent of the normal population falls within (that is, 95% prediction interval).[4] This is most often determined by collecting data from vast laboratory values.

Various medical conditions in pregnancy and non-pregnant states may be traced to abnormal levels of serum calcium.[4] Such conditions include pregnancy-induced hypertension, pre-eclampsia/eclampsia, intra-uterine growth restriction, low birth weight, pre-term birth, lactational difficulty and osteoporosis later in the life.[5] Plasma concentration of calcium usually depends on the net balance of bone mineral deposition and resorption, intestinal absorption and renal excretion under the regulation of parathyroid hormone (PTH), calcitonin and 1,25-dihydroxyvitamin D.[4],[5] Assessing the total calcium level is part of a routine health screening included in the comprehensive and basic metabolic panels used to measure both ionised and bound calcium.[5] Measurement of the total calcium alone, however, may sometimes be misleading, since this value may change without alteration in the ionised calcium concentration. Therefore, this study set out to assess the reference interval of ionised calcium (iCa), as the primary end point as its variation is usually more representative of the general abnormal state. The secondary end points were the determination of the reference interval of total calcium (TCa) and albumin. Variation in calcium levels as seen in pregnancy may be a reflection of the pre-pregnancy level and not necessarily due to the usual normal physiological changes of pregnancy as observed in the second and third trimester of pregnancy.[6] Establishing the reference interval of calcium in a cohort such may allow the strict implementation of the WHO recommendation which stated that, in areas where dietary calcium intake is low, calcium supplementation during pregnancy is recommended for the prevention of pre-eclampsia in all women, especially in those at high risk of developing pre-eclampsia.[7] Omonua et al. demonstrated that administering calcium to a segment of pregnant women significantly reduced the occurrence of pregnancy-induced hypertension[8] raising suspicion of low levels of serum calcium among such women. This, therefore, heralded the need for studies to determine the serum calcium reference interval among women of reproductive age in our locality. Various medical conditions in pregnancy and non-pregnancy state may be traced to abnormal levels of serum calcium. There is no established local reference interval for the Nation's Capital Community to the best of researchers' knowledge. Inferences such as hypo or hypercalcaemia may only be more appreciated if the local reference intervals are known. The current reference intervals used are those obtained from international communities. Therefore, the determination of serum calcium reference interval among women of reproductive age in our subregion becomes pertinent. This will help to implement calcium supplementation to our pregnant women as enshrined in one of the WHO recommendations.

  Materials and Methods Top


The study was given ethical approval by the FCT Human Research Ethics Committee on 23 February 2016. The protocol approval number is FHREC/2016/01/12/23-02-16. The participants were assured that participation was not associated with any risk apart from the slight pain from the prick of the needle. Participation was voluntary and their confidentiality was ensured by labelling the samples with codes to conceal their identities. Informed consents were obtained from them after enrolment.

Study design

Selection and description of participants

This was a cross-sectional study involving 245 women of reproductive age attending 6 primary health-care (PHC) facilities in Abuja, Nigeria. The participants were selected using multistage sampling technique. In the first stage, 2 area councils were selected from the 6 area councils in the federal capital territory (FCT) by convenience sampling method. In the second stage, 3 PHCs were selected from each of the 2 area councils. In the third stage, the sample size was proportionately distributed among the 6 PHCs.

Selected subjects were those women within the reproductive age group (15–49 years) and without pre-existing medical conditions such as chronic hypertension, not on any form of calcium supplementation and had no bone metastasis of carcinomas. Women on tamoxifen, thiazide and those who were pregnant were also excluded.

The study period extended from March 2016 to August 2016.

The primary outcome measure was the determination of the reference interval of free (ionised) serum calcium among women of reproductive age. The secondary outcome measures were the determination of reference intervals of TCa and serum albumin.

Sample size determination

The non-parametric method of sample size determination as recommended by the Clinical Laboratory Standard Institute (CLSI) and International Federation of Clinical Chemists guidelines was used.[9] Using the non-parametric method, it is not possible to distinguish between two percentiles of a distribution that are P% apart, unless at least n = (100/P) − 1 observation have been obtained. To estimate the 2.5th percentile distinct from the 5th percentile or the 95th percentile distinct from the 97.5th (i.e., P = 2.5), a minimum of 39 measurements are required. However, it is undesirable to rely on the extremes of a set of observed values to derive a non-parametric 95% reference interval.[10] Reed et al. suggested that a minimum of 120 observations be secured, one from each subject.[11] Hence, 120 observations are the minimum number of samples that allows the determination of both a 90% confidence interval the reference limits and the central 95% distribution determination. As a standard for general practice, the Clinical Laboratory Standards Institute CLSI recommended a minimum of 120 reference subjects.[9],[12],[13] It is also important to note that, the larger the sample size, the higher the confidence interval and consequently the lesser the bias (for example, for a 95% confidence interval, a minimum of 198 samples are needed). It was based on the foregoing, with a 95% confidence interval as our set goal that we utilised sample size of 245.

Technical information

Their blood samples were collected using serum separator tubes (BD Vacutainer SSTR) which are sample bottles with a vacuum, facilitating collection of pre-determined volume of blood and contained a gel that separated blood cells from serum. The vacutainer also ensured anaerobic environment, absence of which could affect the PH of the blood samples and invariably the ionised calcium levels. The blood samples were collected without a tourniquet with the participant in the supine or sitting position after resting and breathing normally for at least 10 min. There was no muscular activity in the sampling upper limb during sample collection. Following blood collection, the SST vacutainer was inverted 5 times and allowed to stand for 30 min to ensure complete clot retraction. The samples were then centrifuged using a fixed angle centrifuge of 1200 relative centrifugal force (rcf) for 15 min and each serum was decanted into plain a bottle. The serum PH, ionised calcium and total calcium were measured using ionised selective electrodes (Stat-profile PrimeR-Novabiomedical), while serum total calcium, protein and albumin were assayed spectrophotometrically (SpectronicR 20D). The samples were run in batches, i.e., those collected on the same day were analysed together on the same day.

Serum total calcium was determined by using the bromocresol green (BCG) method, wherein albumin reacts with BCG gives a purple-coloured complex whose absorbance is read at 630 nm in the spectrophotometer. The protein was determined by using the biuret method.


The data on sociodemographic characteristics were analysed using SPSS version 20. Five participants were eventually excluded from the analysis due to obesity and underweight respectively. This decision to exclude the aforementioned was predicated on reported findings on the observations of effects of obesity and underweight on calcium homeostasis and PTH function.[14],[15],[16] These exclusions after recruitment left a total of 240 subjects who underwent the total assessment including the bio-specimen analysis.

  Results Top

A total of 245women were recruited into the study from six primary health centres of two area councils within the FCT but, 5 participants were eventually excluded from analysis due to obesity and underweight respectively. The mean age of the study subjects was 29.2 ± 8.3 years with a median body mass index (BMI) of 23.6 kg/m2 [Table 1]. All the subjects had a blood pH range within the physiologic range (7.35–7.45) with the mean pH as 7.38. [Table 2] shows the classification of patients according to their sociodemographic characteristics: Majority were traders (50.4%), while the least were farmers (6.3%). About 11% of the study population had no formal education. The mean serum ionised calcium amongst the study participants was 1.08 mmol/L (±0.19). Using the non-parametric method of reference interval determination as recommended by the Clinical and Laboratory Standards Institute (CLSI), the reference interval for ionised calcium (iCa) was 0.88–1.4 mmol/L, corresponding to 2.5th percentile (rank value number 6) and 97.5th percentile (rank number 235), respectively. This range equates to 95% confidence limits and on categorisation of the reference interval into seven subgroups [Table 3]. The reference interval for TCa and corrected total calcium (CorrTCa) were 2.18–2.82 mmol/L and 2.06–2.72 mmol/L, respectively [Table 4]. The reference intervals for total protein and albumin were 5.7–9.4 mg/dl and 3.3–5.2 mg/dl respectively [Table 4].
Table 1: Distribution of age, body mass index and blood pressure

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Table 2: Classification of patients according to their sociodemographic characteristics

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Table 3: Subgroup categorisation of reference interval for serum ionised calcium

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Table 4: Reference intervals of other measured parameters

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  Discussion Top

In this study, the mean serum ionised calcium (iCa) obtained was 1.08 mmol/L. This was slightly lower than 1.22 mmol/L and 1.29 mmol/L reported by previous authors.[17],[18] The reference interval of ionised calcium (iCa) in this study was 0.88–1.4 mmol/L. The lower limit obtained was lower than those from other studies from developed countries like Sweden with their lower limit values ranging from 1.03 mmol to 1.10.[19],[20] These marginal differences could be related to factors such as study participants, method of laboratory analysis and specimen collection procedures as these have been proven to affect outcomes.[21],[22] In this study, the reference interval for CorrTCa (2.06–2.72 mmol/L) was closely related to the findings of Adewolu from Benin city (2.03–2.63 mmol/L).[23] This is despite the fact that we had not lost sight that the values from Adewolu's study had no adjustment of the measured total Ca and the sample size was much smaller (n = 60). Unfortunately, the various secondary level health-care institutions that these study primary health centres (PHC) site fed, serum ionised calcium is not assessed and thus there are no values to use for comparison with our findings. In addition, TCa which is assessed at the tertiary health facilities and general hospitals within the FCT is based on a reference interval of 2.2–2.6 mmol/L) that was not generated locally but from the manufacturer's inserts in laboratory kits. More often than not, the values enclosed in these kits are probably products of studies conducted in a different race and environment. This operational reference interval differs from the outcome of our study findings, especially in the upper limit (2.6 vs. 2.82 mmol/L) by 0.22 mmol/L and this could have implications in clinical practice. It is pertinent here to further stress that, as little as this difference in value may appear in determining the reference interval, it may affect the resultant clinical outcome. Suffice to state that the variations in protein binding of calcium (mainly albumin) could be the basis of these deviations in the levels of serum total calcium.[24],[25] This is further simplified that, as 80% of the bound calcium present in the circulation is carried by Albumin, changes in serum albumin concentration trigger significant changes in the concentration of total calcium. Hypoalbuminemia is the most common cause of pseudohypocalcaemia.[24],[25] One drawback of the study is that the iCa values were not adjusted for pH since pH is known to affect iCa levels. However, it suffices to state that, the effect of pH would not, expectedly, have had a significant effect on the study findings given that the pH of all study participants was measured and all were within the physiologic range. In addition, the pH effect on iCa is only of major clinical significance in cases of acidosis or alkalosis.

  Conclusion Top

This study has established the nomogram of the reference interval of iCa as 0.88–1.4 mmol/L and the mean serum total calcium level as 2.5 mmol/L among women of reproductive age in FCT, Nigeria. The lower limit of this normal range is about 0.22 lower than the operational kit related values in the community. The accompanying reference intervals for total protein and albumin were 5.7–9.4 and 3.3–5.2 mg/dl, respectively.


The established reference intervals above from this study should be employed as operational values in all hospitals within FCT as these had a noticeable difference with values provided by kits manufacturers. A nation-wide study to establish the national and or regional reference interval of serum iCa, Total corrected calcium, protein and albumin is recommended to domicile values for our various communities and to informed guideline policy changes. A study to determine the dietary content of calcium is also advocated.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

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Abbassi-Ghanavati M, Greer LG, Cunningham FG. Pregnancy and laboratory studies. Obstetr Gynecol 2009;114:1326.  Back to cited text no. 3
Stephen KB, William JM, Mashall WL. Clinical Biochemistry: Metabolic and Clinical Aspects. Philadelphia: Churchill Livingstone/Elsevier; 2008.  Back to cited text no. 4
Daniels R. Delmar's Guide to Laboratory and Diagnostic Tests. 2nd ed. Boston, MA: Cengage Learning; 2010.  Back to cited text no. 5
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CLSI. Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved Guideline – Third Edition. CLSI Document EP28-A3c. Wayne, PA: Clinical and Laboratory Standard Institute; 2008.  Back to cited text no. 9
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Solberg HE. Approved recommendation on the theory of reference values (1987). Part 5. Statistical treatment of collected reference values Determination of reference limits. J Clin Chem Clin Biochem 1987;25:645-56  Back to cited text no. 12
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Ren XH, Yao YS, He LP, Jin YL, Chang WW, Li J, et al. Overweight and obesity associated with increased total serum calcium level: Comparison of cross-sectional data in the health screening for teaching faculty. Biol Trace Elem Res 2013;156:74-8.  Back to cited text no. 14
Coin A, Sergi G, Benincà P, Lupoli L, Cinti G, Ferrara L, et al. Bone mineral density and body composition in underweight and normal elderly subjects. Osteoporos Int 2000;11:1043-50.  Back to cited text no. 15
Kamycheva E, Sundsfjord J, Jorde R. Serum parathyroid hormone level is associated with body mass index. The 5th Tromsø study. Eur J Endocrinol 2004;151:167-72.  Back to cited text no. 16
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Boink AB, Buckley BM, Christiansen TF, Covington AK, Maas AH, Müller-Plathe O, et al. Recommendation on sampling, transport, and storage for the determination of the concentration of ionized calcium in whole blood, plasma, and serum. IFC Scientific Division, Working Group on Ion-Selective Electrodes (WGSE). J Int Fed Clin Chem 1992;4:147-52.  Back to cited text no. 21
Redmond J, Jarjou LM, Zhou B, Prentice A, Schoenmakers I. Ethnic differences in calcium, phosphate and bone metabolism. Proc Nutr Soc 2014;73:340-51.  Back to cited text no. 22
Adewolu OF. Serum sodium, potassium, calcium and magnesium in women with pregnancy induced hypertension and preeclampsia in Oredo local Government, Benin Metropolis: A pilot study. Afr J Med Health Sci 2013;12:1-5.  Back to cited text no. 23
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  [Table 1], [Table 2], [Table 3], [Table 4]


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