The present study was a cross-sectional analytical study conducted on blood donors presenting to the Blood bank in the Department of Transfusion Medicine, Government Medical College Kottayam, Kerala, India, between 31^st December 2016 to 31^st December 2017, after obtaining the approval from Institutional Review Board (IRB No.126/2016).

Sample size calculation: The sample size was calculated, as was suggested by Mittal R et al., [1].

The calculation came out to be 132, where N=sample size, Z=1.96 at 5% level of significance, SD=standard deviation: 25.85, d=absolute precision; 17% of SD. Considering 10% non-response, sample size was taken as 150.

Inclusion criteria: All donors between the age group of 18-40 years during one year period and weighing more than 45 kg were included in the study after obtaining the informed consent.

Exclusion criteria: Donors with Hb <12.5 g%, or those with history of smoking, alcoholism, systemic illness like diabetes and hypertension or with history of blood loss in the past one year by surgery/accident or medical conditions like menorrhagia, donors with liver disease, malignancy, inflammatory conditions, thyroid disorders and the ones unwilling to participate in this study were excluded from the study.

The total sample of 150 selected donors, and all relevant details- demographic details, history of smoking and alcohol intake were noted. In the present study, the total sample was divided into four groups according to the number of donations per year-group I were the first time donors with no previous history of blood donation, Group II were the donors who had previously donated once in the previous year, group III were the donors who had donated twice in the previous year, group IV were the donors who had donated thrice in the previous year [Table/Fig-1].

A six mL of whole blood was collected from each donor during process of blood donation. Out of six mL of the blood, two mL was collected in Ethylene Diamine Tetra Acetic Acid (EDTA) tubes which were used for estimating Hb, PCV, MCV, MCH, MCHC while four mL collected in plain tubes for serum ferritin analysis.

These test were performed in Sysmex XS-1000i haematology analyser on whole blood anticoagulated with potassium EDTA. Haematocrit (HCT) is measured as the ratio of the total Red Blood Cell (RBC) volume to whole blood using cumulative pulse height detection. Hb is converted to Sodium Lauryl Sulfate (SLS)- Hb and read photometrically.

SLS is the RBC lysing reagent that releases the Hb to be measured by SLS Hb method. Its active ingredient was Sodium Lauryl Sulfate 1.7 g/L and is stored at controlled temperature of 2-30°C, with stability for 60 days.

Reagents used: Sysmex Cellpack Ethanol Extract of Pinus Koraiensis Leaves (EPK) with ingredients Sodium Chloride 6.38 g/L, Boric Acid 1.00 g/L, Sodium Tetraborate 0.20 g/L and EDTA-2K 0.20 g/L, Stored at a controlled temperature of 5-30°C, was used as whole blood diluent for use in the determination of Hb and impedance counting and sizing of blood cells. It also forms a laminar sheath flow around the diluted sample for hydrodynamic focusing of the RBCs and platelets.

Sysmex stromatolyser- Differential Lysing Reagent 4DL Product Code by Manufacturer FFD-200A (FFD) is the lysing reagent and diluent for the enumeration of neutrophils, lymphocytes, monocytes, eosinophils, and basophils after eliminating RBC stroma. Its active ingredients include non-ionic surfactant 0.18% and organic quaternary ammonium salts 0.08% and is stored at a controlled temperature of 2-35°C, with a stability for 60 days.

Dying Solution 4DS Product Code by Manufacturer FFS-800A (FFS) is used to stain leukocytes in diluted, lysed blood samples for the determination of the 5-part differential including neutrophils, lymphocytes, monocytes, eosinophils and basophils. Its active ingredients were ethylene glycol 96.9%, methanol 3.0% and polymethine dye 0.002% and were stored at controlled temperature of 3-35°C with stability for 90 days.

Calibration: Calibration is done using Sysmex Sysmex Corporation System (SCS)-1000 Calibrator. The ranges considered as reference for this study were: Hb-13.6-16.7 g/dL in males and 11.8-15.1 g/dL in females, PCV-41-50% in males and 36-46% in females, MCV- 82-99 fL, MCH-27-32 pg and MCHC-32-36 g/dL.

Four mL of blood of each donor was collected in plain tubes and serum was separated by centrifugation at 3000 rpm for 10 minutes and was kept at -20°c. The sample underwent CLIA by Beckman Coulter Access 2 Immunoassay system. Chemiluminiscence is the emission of light when an electron returns from an excited or higher energy level to a lower energy level. The excitation event is caused by a chemical reaction; caused by oxidation of an organic compound.

Reagent used: Access ferritin reagent containing paramagnetic particles coated with goat antimouse IgG: mouse antiferritin complexes, Bovine Serum Albumin (BSA) in Reagent 1a (R1a) and Goat antiferritin alkaline phosphatase (bovine) conjugate, BSA, protein (goat, murine) in R1b.

Calibration: Calibration is done using access ferritin calibrator with following references:

S0: buffered BSA matrix

S1-S5: human liver ferritin, buffered BSA matrix

S0-0 ng/mL, S1-10 ng/mL, S2-50 ng/mL, S3-200 ng/mL, S4-500 ng/mL, S5-1500 ng/mL.

Iron stores were considered normal at serum ferritin values from 23.9-336 ng/mL in males and from 11-307 ng/mL in females, hence these were considered as reference ranges for the present study.

Statistical data was entered in Microsoft Excel and statistical analysis was performed in SPSS version 16.0. Microsoft Word and Excel were used to generate tables. All the variables were analysed and compared between the four groups of study sample. Continuous data are reported as mean±Standard Deviation (SD). ANOVA test was performed to find association between frequency of blood donation and blood parameters. Pearson’s correlation coefficient was used to find association between serum ferritin levels and other blood parameters. Significance is assessed at 5% level and a p-value <0.05 was considered as statistically significant.

Mean age of the study population was 27.53±6.873 years. (Range: 18-40 years, with majority (95.3%) being males [Table/Fig-2]. Males showed a mean ferritin level of 88.351 mg/mL and females showed a mean ferritin level of 26.686 ng/mL. All the study participants were taking a non-vegetarian diet and none gave the history of major surgery/blood loss in the past one year. None among the females had child birth in the past two years and none reported menorrhagia. Their past histories regarding smoking and alcoholic habits is shown in [Table/Fig-2].

Among those who donated blood more than once in the past one year (n=113), mean interval between the donations was 5.22±1.450 months. The mean values of laboratory investigations and blood parameters of donors is shown in [Table/Fig-3].

Mean serum ferritin value for donors (first, second, third and fourth) is shown in [Table/Fig-4]. There was no significant correlation between serum ferritin level and frequency of blood donation in this study.

The p-value of MCH, MCHC is less than 0.05 and showed significant association with frequency of blood donation. Hb, PCV, MCV did not show any significant association with frequency of blood donation [Table/Fig-5].

Pearson correlation coefficient was used to compare serum ferritin levels with other blood parameters. Low positive correlation was seen between Hb, PCV with serum ferritin levels. This was found to be statistically significant with a p-value <0.001. Though negligible correlation was seen between serum ferritin and MCV, MCH, this was found to be statistically significant [Table/Fig-6].

The present study compared serum ferritin levels with frequency of blood donation, Hb levels and other blood indices (MCV, MCH, MCHC). They showed a mean serum ferritin level of 85.473 ng/mL. This study shows that there was no significant relation between frequency of blood donation and serum ferritin levels. This was in contrast to studies conducted by Mittal R et al., and Mahida VI et al., [1,21]. Considering distribution of study population based on number of blood donations during life time 37 donors who were first time donors, had a mean ferritin level of 82.581 ng/mL. The present study did not showed any significant relation with total number of blood donations during life time. This was in contrast to the study conducted by Simon TL et al., which showed that 8% of men and 38% of women have reduced iron stores, as assessed by serum ferritin status, after five donations [22]. In the study by Milman N and Kirchhoff M, 26% of regular blood donors had low ferritin levels and 12% were found to be anaemic [11]. In Nigeria, in 1990, Usanga EA observed that the mean ferritin concentration of 64.75±4.6 ng/mL in first time donors was significantly higher than the mean value of 49.19 ng/mL among frequent blood donors suggesting that some blood donors may have pre-latent or latent iron deficiency at the time of donation and become iron deficient after blood donation [23].

In Adediran A et al., study the mean serum ferritin level was significantly lower in the regular donors than in the first time donors [24]. Szymczyk-Nuzka M and Wołowiec D when determining the prevalence of iron deficiency in regular blood donors noted a lower serum ferritin in this group in contrast with first time donors who had normal serum ferritin levels [25]. Norashikin J et al., also reported significantly lower serum ferritin levels in regular blood donor [26]. Several investigators have noted a significant reduction in ferritin levels with increasing donations [27-29]. Contrary to these published studies, this study did not show any significant association between serum ferritin levels in first time donors and regular donors.

Though MCH, MCHC showed statistically significant relation with frequency of blood donation, they are not clinically relevant. Group 1 donors showed a mean MCH of 28.273 fL and Group 4 donors showed a mean MCH of 27.889 fL. Similarly, Group 1 donors showed a mean MCHC of 33.78 g/dL and group 4 donors showed a mean MCHC of 33.53 g/dL. These findings correlated with study conducted by Tailor HJ et al., [5]. There was a significant correlation between the frequency of donations, last donation interval and blood indices in the study conducted by Tailor HJ et al., [5]. A positive correlation was seen between Hb and ferritin levels and PCV and ferritin levels. This was in correlation with study conducted by Suominen P et al., [30]. Also, a significant correlation was seen between MCV and ferritin levels and MCH and ferritin levels. This similar finding is obtained in study conducted by Abdullah SM [6].

Considering distribution of study population based on gender 95.3% were males and 4.7% were females. Males showed a mean ferritin level of 88.351 mg/mL and females showed a mean ferritin level of 26.686 ng/mL. Here, there was no statistically significant relation between frequency of blood donation and serum ferritin levels. In the study conducted by Deepa DP et al., distribution on the basis of number of donations per year and serum ferritin <15 ng/mL in male donors were 6.9 % in first time, 19.4 % in once a year, 26.7% in twice a year and 50% in thrice year donation [31]. Among female donors 40.7% in first time, 50% in once a year, 50% in twice a year donation had serum ferritin levels <15 ng/mL and hence there was a definite correlation between dwindling of serum ferritin level and the frequency of donation [31].

Also, sample size was 150 as compared to 400 in the study conducted by Mittal R et al., [1]. Rosvik AS et al., recommended that donors with serum ferritin levels below 50 μg/L should be given iron while donors with levels above 80 μg/L needs no additional iron [32]. There are no formal recommendations in standards or regulations for the use of iron supplementation after blood donation, but dietary advice is a relatively widespread practice in donor centers. Even casual use of iron supplementation has shown to be advantageous to blood donors [33]. The blood donors at risk of developing iron deficiency can be detected only by the estimation of the levels of serum ferritin levels. The iron status of the donors should be identified and necessary steps for iron supplementation need to be taken to fulfill the national and global drive for the blood requirements.

Here, contrary to previous studies, a statistically significant correlation was not obtained between serum ferritin levels and frequency of blood donation. This may due to difference in socio-economic status, diet, sample size etc. All the donors belonged to Kottayam district of Kerala, middle class or higher middle class family consumed red meat most days of the week and made sample size to 150. Also, total number of life donations was less than 10 in majority of the donors. So, this study shows that depletion of iron stores is not a concern for low frequency donors and blood donation should be encouraged in our society.

In this study, there was no statistically significant relation between Hb levels and frequency of blood donation. This was similar to the study by Abdullah SM, Finch CA et al., and Yousefinejad V et al., [6,13,34]. In the study, by Milman N and Kirchhoff M, the study population was large and they have categorised the ferritin levels as decreased and depleted [11]. Study done by Norashikin J et al., took into consideration a period of two years for assessing the frequency of donation [26], whereas, in this study the groups were categorised using one year for the frequency of blood donation.

In the study, conducted by Mahida VI et al., there was no significant difference between ferritin levels in controls and donors donating less than 20 units [21]. Akpotuzor J et al., reported that there was no observable difference in biochemical iron parameters between regular donors and normal controls [35]. This was similar to the results obtained in this study. In contrast, several other studies have documented lower serum ferritin levels in regular blood donors compared with healthy controls [31,32].

In the present study, iron stores were estimated based on serum ferritin levels. Other parameters indicating iron status such as, serum iron, transferrin saturation, Total Iron Binding Capacity (TIBC) were not included. It was a small sample study with all subjects belonging to Kottayam Kerala, specifically, which might be a factor affecting measurement of blood indices.

Blood donation is an act of benevolence rather than altruism. Both the donor and recipient gains. The study showed no significant relation between frequency of blood donation and serum ferritin levels and positive correlation between serum ferritin levels and Hb, MCH, MCHC showed significant relation with frequency of blood donation. The study data suggested that ferritin levels, Hb concentration, PCV are not significantly affected by regular blood donation. A larger study may be needed to confirm these preliminary findings. This study shows that a person can donate blood three times a year without being concerned about depletion of iron stores and blood donation should be encouraged in our society.