Establishment of normal reference range of serum hepcidin in Indian blood donors

Abstract

BACKGROUND: 

Hepcidin-25, a polypeptide hormone, plays a major role in iron metabolism and is found to be reduced during iron deficiency; therefore, testing for hepcidin can be utilized as an indicator of bioavailability of iron. Globally, reference range values for hepcidin have been established in different communities. The aim of the present study was to establish the normal reference range values of serum hepcidin in Indian blood donors to fathom the baseline and reference point of hepcidin.

MATERIALS AND METHODS: 

A total of 90 donors fulfilling the eligibility criteria were recruited in the study consisting of 28 males and 62 females. Blood samples collected were used to execute hemoglobin (Hb), serum ferritin, and hepcidin assays. Serum hepcidin-25 isoform was detected by a commercial competitive enzyme-linked immunosorbent assay kit according to the manufacturer's instructions. Hb and ferritin were evaluated by the standard methods.

RESULTS: 

The mean ± standard deviation (SD) of Hb level in males was 14.62 ± 1.34 g/dL and females was 13.33 ± 0.76 g/dL. The mean ± SD of ferritin level in males was 113 ± 56.12 ng/mL and females was 62.65 ± 40.8 ng/mL. Similarly, the mean ± SD of hepcidin level in male donors was 22.18 ± 12.17 ng/mL and female donors was 10.95 ± 6.06 ng/mL. The established reference range values of Hepcidin were 6.32–46.06 ng/mL for males and 3.44–24.78 for females.

CONCLUSION: 

These findings suggest that further studies with larger groups of donors are mandatory to produce reference values of hepcidin that can be précised to the whole populace in India.

Introduction

Hepcidin, a 25 amino acid polypeptide hormone, synthesized in the liver, regulates iron balance by systemic iron metabolism in the body.[¹] It is synthesized by hepatocytes and decreases iron absorption in the duodenum and release of iron from macrophages, through binding to ferroportin consequently controlling the plasmatic concentration of the iron and its storage.[²] Therefore, testing for hepcidin can be used as an indicator of bioavailability of iron. Hepcidin can be detected either by mass spectrometry or by immunological methods. Detection by mass spectrometry is technically challenging and provides information about different isoforms of hepcidin.[³⁴] The immunological methods are more common and currently in practice for detection although hepcidin is not usually done as a routine diagnostic parameter. Frequently used parameters to detect the iron status are serum ferritin, iron, transferring, total iron-binding capacity (TIBC), transferrin saturation (iron/TIBC), and C-reactive protein levels, but each one of these has constraint,[⁵] hence to initiate this marker as a routine diagnostic parameter establishment of normal reference range is essential.

Globally, reference range values for hepcidin have been established in different communities; Pasricha et al.[⁶] reported it as a diagnostic test in healthy premenopausal women in Australia with reference range values of 5.4 ng/mL–174.6 ng/mL; similarly, Galesloot et al.[⁷] also established normal reference ranges for hepcidin according to gender and age in echelon of 5 years each in the general population of The Netherlands.

Sdogou et al.[⁸] have reported it from Greece pediatric populace and established serum hepcidin reference range measurements in healthy children and evaluated differences among age groups with the help of immunoassay-based technique. The more recent hepcidin reference range was reported by Gonzo et al.[⁹] in the year 2017 in Namibian blood donor populace. The values established in their study were 17.186–91.237 ng/mL for females and 18.227–81.541 for males. Emphasis on criteria for the selection of blood donors and management of donation-induced iron depletion is a major concern for clinicians.[¹⁰] As blood donors are found to be at high risk of iron deficiency,[¹¹] hence, this populace is apt to assess novel tests of iron deficiency, and for prognosis and accurate diagnosis of any disease, appropriate reference range values are essential.

The present study was carried out to establish normal reference range values of serum hepcidin in Indian blood donors which might be supportive to figure out the baseline and reference point of hepcidin and to compare them with previously established reference values elsewhere which will help to explore its quantitative characteristics.

Materials and Methods

Study population

The study was conducted at the Department of Transfusion Medicine, in a tertiary care hospital, North India. The study was approved by the Institutional Ethics Committee. Ninety normal healthy voluntary blood donors of either sex, given consent to participate in the study, were recruited to establish reference ranges. Healthy individuals were defined according to the donation criteria as set out by the Ministry of Health and Family Welfare, Government of India, in the year 2017. The major criteria for donation include.

• First-time blood donors (because serum hepcidin is inversely proportional to number of blood donations)[¹²]
• Donor shall be in good health, mentally and physically fit
• Donor should be between the age of 18 and 65 years
• Weight of the donor should be ≥45 kg with hemoglobin (Hb) ≥12.5 g/dL
• Donation interval should be more than 90 days for males and 120 days for females
• Donor shall be free from any disease which can be transmitted by blood transfusion
• Physiological status of women for blood donation should be:
• Defer for 12 months after delivery and 6 months after abortion
• Defer for total period of lactation and period of menstruation.

We have examined the participant's health and medical history by using standard donor questionnaire, comparable to Gonzo et al.[⁹] They have also used a questionnaire to carry out the physical evaluations of their study participants to assist in determining their health status and lifestyle. Earlier, Galesloot et al.[⁷] in their study in a large, well-phenotyped sample of the general population to determine the reference range of hepcidin have asked all the participants to fill a questionnaire to establish their health status, lifestyle, and medical history.

The standard donor questionnaire consists of different physical examination measures that include examining imperative signs such as blood pressure, weight, body mass index (BMI) as well as query about general health status of the donor including history of anemia and chronic illness. The purpose of donor questionnaire is to assess participants’ general health and medical history on the day of collection of blood to allow them to donate. They should meet certain criteria as mentioned by the Ministry of Health and Family Welfare, Government of India, in the year 2017 to qualify to donate blood.[¹³]

Sample size calculation

Sample size at 90% power for females

Sample size is calculated on the basis of variation in hepcidin level among the study cases using the formula:

where σ = 17.19, the standard deviation (SD) of the hepcidin level in females among the study cases:

• d = 15% of mean hepcidin level, the difference considered to be statistically significant[⁹]
• Type I error α =5% corresponding to 95% confidence level
• Type II error β =10% for detecting results with 90% power of the study.

Hence, the required sample size was calculated to be:

n = 62.

Sample size at 80% power for males

Because of less availability of first-time male donors, sample size was calculated at 80% power on the basis of variation in hepcidin level among the study cases using the formula:

where σ = 16.49, the SD of the hepcidin level in males among the study cases

• d = 20% of mean hepcidin level, the difference considered to be statistically significant[⁹]
• Type I error α = 5% corresponding to 95% confidence level
• Type II error β = 20% for detecting results with 80% power of the study.

Hence, the required sample size was calculated to be:

n = 28.

Laboratory methods

After filling the questionnaire and obtaining the participants’ weight and vital signs such as blood pressure, blood samples (venous) were collected from healthy blood donors after obtaining consent. Samples collected were used to execute Hb, serum ferritin, and hepcidin assays. The samples for these assays were collected simultaneously along with blood donation in order to evade deviation in the concentration of hepcidin. After the blood donation, hepcidin concentration might get affected due to decrease in blood volumes which causes subsequent decline in iron content.[¹⁴]

Hepcidin is reported to be the major iron-regulatory hormone[¹⁵] which undergoes diurnal variation;[⁷¹⁶] considering this, all blood samples were collected between 08.00 a.m. and 9.00 a.m.

Serum hepcidin-25 was diagnosed according to the manufacturer's instructions by using a commercially available competitive enzyme-linked immunosorbent assay (ELISA) kit (Hepcidin-25 (bioactive) HS ELISA; DRG Instruments GmbH, Marburg, Germany). This ELISA kit is a solid-phase ELISA, based on the principle of competitive binding with a dynamic range of 0.135–81 ng/mL, mean intra-assay precision: 5.5%, mean inter-assay precision: 12.5%, and analytical sensitivity: 0.135 ng/mL.

ELISA plates containing 96 μl wells were precoated with a monoclonal (mouse) antibody directed toward the antigenic site of the bioactive hepcidin-25 molecule. Endogenous hepcidin-25 of a subject sample contends with the added hepcidin-25 biotin conjugate (enzyme conjugate) for binding to the precoated antibody. After incubation, the unbounded conjugate is washed off. Binding of the enzyme conjugate is detected by the streptavidin peroxidase enzyme complex. Color is developed by the addition of substrate solution and is stopped up after a short incubation. The intensity of color developed is inversely proportional to the concentration of hepcidin-25 present in the serum sample.

The other two hematological parameters, Hb and ferritin, were evaluated by Medonic M20 (Boule Medical AB, Sweden) and COBAS e 411 (Roche Diagnostics GmbH, Germany), respectively. Donors having Hb concentration <12.5 g/dL and ferritin level <12 ng/ml were excluded from the study.

Statistical analysis

The results were analyzed by descriptive statistics. Categorical data were summarized in proportions and percentages (%) while discrete as mean and SD.

The different statistics methods employed in the present analysis were:

The arithmetic mean

The most widely used measure of central tendency is arithmetic mean, usually referred to simply as the mean, calculated as:

The standard deviation (σ)

It is calculated by using the formula:

Prediction and confidence interval

Prediction intervals predict the value of an individual which lies inside the group. The key element is that the prediction interval describes about the distribution of values, not the unpredictability in establishing the population mean.

The 95% prediction range of biochemical parameter is calculated by using the formula:

The 95% confidence range of biochemical parameter is calculated by using the formula:

where x (bar) is the sample mean, s is the sample SD, and n is the sample size.

The P value was taken significant when < 0.05 (P < 0.05) and confidence interval of 95% was taken.

Results

A total of 90 donors were recruited for the study after the stratification according to their eligibility to donate blood. It consists of 28 males and 62 females. The mean age of study subjects was 31.87 ± 9.01 years. The mean age of males was 29.86 ± 7.12 years while the mean age of females was 32.77 ± 9.66 years. Table 1 depicts the donor profile and their iron parameters. The mean ± SD of Hb level in male donors was 14.62 ± 1.34 g/dL and female donors was 13.33 ± 0.76 g/dL. The mean ± SD of ferritin level in total population was 78.37 ± 51.72 ng/mL; in male donors, it was 113 ± 56.12 ng/mL; and in female donors, it was 62.65 ± 40.8 ng/mL. Similarly, the mean ± SD of hepcidin level in total population was 14.44 ± 9.9 ng/mL; in male donors, it was 22.18 ± 12.17 ng/mL; and in female donors, it was 10.95 ± 6.06 ng/mL.

Table 1: Summary of donors’ profile and iron parameters (mean±standard deviation)

Data were analyzed and reference range was estimated using prediction interval method. Table 2 shows the descriptive summary and reference range estimation of hepcidin level among males and females; the mean hepcidin level of study subjects was 22.18 ± 12.39 ng/mL. This provides the 95% confidence range for mean hepcidin level as lower bound 17.59 ng/mL and upper bound 26.77 ng/mL, and the 95% prediction range which can be taken as reference range is estimated to be lower bound 0.00 and upper bound 46.89 ng/mL. Hepcidin distribution was with skewness index of 1.10 and kurtosis index of 0.82 which might be due to the comparatively small number of study participants. While, among females, the mean hepcidin level of study subjects was 10.94 ± 6.11 ng/mL. This provides the 95% confidence range for mean hepcidin level as lower bound 8.68 ng/mL and upper bound 13.21 ng/mL, and the 95% prediction range which can be taken as reference range is estimated to be lower bound 0.00 and upper bound 23.02 ng/mL. Hepcidin distribution was with skewness index of 1.10 and kurtosis index of 0.82 which again might be due to the comparatively small number of study participants.

Table 2: Descriptive summary and reference range estimation of hepcidin level using prediction interval method among males and females

Reference range of hepcidin level was also estimated using percentile method [Table 3]. Among males, the 5^th and 95^th percentiles of hepcidin level were found to be 6.32 units and 46.06 units, respectively, which provides the 90% percentile range for hepcidin level as 6.32–46.06 and can be taken as a reference range. While, among females, the 5^th and 95^th percentiles of hepcidin level were found to be 3.44 units and 24.78 units, respectively, which provides the 90% percentile range for hepcidin level as of 3.44–24.78 and can be taken as a reference range.

Table 3: Descriptive summary and reference range estimation of hepcidin level using percentile method

Table 4 shows the comparison between prediction interval and percentile method. The PI method provides larger range as a reference range as compared to percentile method and so includes more normal cases than the percentile method. Total 28 male, 27 (96.4%) were within the range and among female out of total 62, 58 (96.7%) were within the range. Whereas in percentile method only 92.9 % male and 93.3 % female were within the range.

Table 4: Comparison between prediction interval and percentile method

Discussion

In this study, reference ranges of serum hepcidin concentration were established for the healthy, nonanemic Indian blood donor. We demonstrated sex-specific reference ranges of serum hepcidin, and for its calculation, the participants having characteristics which manifestly manipulates the concentration of hepcidin, namely anemia and low ferritin concentration, were excluded; as a result, unambiguously affirmed precise serum hepcidin reference ranges were established,[¹⁷] and these findings are consistent with those reported earlier.[⁶⁷⁸] Study participants have not reported any restriction in their dietary behavior and BMI of all the subjects whether males or females were within normal values.[¹⁸¹⁹] The serum hepcidin reference ranges in our study are in accordance with hepcidin concentrations in healthy controls previously reported.[^20212223] The variance in absolute hepcidin level evident globally is due to the different assay methods; a comparative study on different assay methods for hepcidin concentration established that although there is a significant difference in the absolute value for results at all laboratories, when compared, the results for samples are showing good correlation and low analytical variation[²⁴] which obviously exemplify the necessity for coordination. Ganz et al.[¹⁶] found a significant difference in serum hepcidin level between men and women; in our study, we also observed a considerable difference among both sexes.

In our study, we found a correlation between hepcidin and ferritin levels; in males, the mean hepcidin level was higher as compared to females so as with ferritin level also; the mean level of ferritin was higher in males as compared to females, indicating an association between serum ferritin and serum hepcidin levels, and these findings are consistently been reported previously.[^16202526] In a very recent study on blood donors from India, researchers have reported that serum hepcidin levels are moderately correlated with serum ferritin levels and stated the effectiveness of hepcidin as a diagnostic marker for iron deficiency.[¹²] They suggested that large multicenter studies are required to attain the final conclusion.

Conclusion

Our study provides sex-specific reference ranges of serum hepcidin concentration in Indian blood donors. In iron-deficient conditions like anemia, usefulness of these ranges is helpful as baseline information for the exposition of the properties of hepcidin-25. In the present study, the established reference range values were 6.32–46.06 ng/mL for males and 3.44–24.78 for females. Development of this index of hepcidin reference range would be helpful in interpretation of clinical samples and facilitate interlaboratory collaborations. The use of an authentic commercial kit assures the consistent outcome. These findings propose further studies with larger groups of donors would be required to produce reference values of hepcidin that can be specified to the entire populace in India.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.