Soluble Transferrin Receptor Levels of Apparently Healthy Adults in Port Harcourt, Nigeria

Background: soluble transferrin receptor is an early marker of tissue iron deficiency before onset of anaemia. sTfR is one of the diagnostic markers for iron deficiency anaemia. Iron deficiency anaemia is one of the most prevalent causes of anaemia in our environment. However, there is no established reference range for this diagnostic marker sTfR in our environment. Objectives: To determine the soluble transferrin receptor levels in apparently normal adults in Port Harcourt Nigeria, determine the reference value of sTfR in the study population. Methods: This is a descriptive cross sectional study conducted at the university of Port Harcourt Teaching Hospital. One hundred and fiftyparticipants who satisfied the inclusion criteria were enrolled for this study. Full blood count and sTfR concentration were assayed on anticoagulated blood samples using a 3-part auto analyzer (Sysmex –KX2IN®) and Human sTfR ELISA kit by BioVendor respectively. The results were analysed using SPSS version 21. P value <0.05 was considered significant. Results: The mean sTfR concentration of the study population was 0.89±0.46ug/mL with a range of 0.33.05ug/ml. The mean value of sTfR for males and females were 0.92±0.49ug/ml and 0.82±0.37ug/ml and the mean sTfR levels did not differ significantly for age and sex. Conclusion: The reference value of sTfR concentration in healthy adults in Port Harcourt was established as 0.3-3.05ug/ml. This study found no statistically significant relationship of sTfR between the different sexes and ages.


Introduction
Iron deficiency anaemia (IDA) is the most common anaemia in the world despite abundance of iron in the environment. The examination of bone marrow aspirate, stained with Prussian blue stain for iron, is still considered as the best method for evaluating iron status in patients with indeterminate laboratory findings. Its draw backs are that it is invasive, expensive, the results may be operator dependent and the aspirate obtained may not be adequate. It has also been shown to be uncomfortable and therefore not practical for routine use. [1,2] Due to the high global burden of iron deficiency anemia which has made it a public health issue, there is a need to devise a screening tool that can be used to detect iron deficiency before onset of anemia to aid early intervention and reduce the mortality and morbidity associated with IDA. This necessitates the need for a less invasive, specific and sensitive means of detecting tissue iron deficiency before onset of anemia. Soluble transferrin receptor (sTfR) assay is a feasible option. [3] Transferrin receptors are expressed on all dividing cells and its rate of synthesis is related to the cell's iron requirement. [4] There are are up to 10,000 -100,000 sTFr molecules per cell in actively dividing cells especially in erythroid precursors in the bone marrow and placental syncythiotrophoblast. sTfR concentration increases up to two folds in iron deficiency, making it an important tool for detecting early tissue iron deficiency before onset of anaemia. The other assays for determining iron deficiency, like serum iron, ferritin, total iron binding capacity, etc, are affected by inflammatory changes. [5] sTfR is not affected by inflammationmaking it superior to these other assays. [6,7] Sweet et al reported that sTfR measurement could replace other assays for iron deficiency state especially in subclinical iron deficiency present in their study population which was undetected by these other assays. [8] There is no difference between sTfR concentrations in healthy males and females, [9,10] but the values are higher in children compared to adults. [11] However, Blacks have a higher sTfR concentration than non-blacks. [12] This difference may be related to the well-known but unexplained difference in haemoglobin concentrations in Blacks and Caucasians.Therefore, misdiagnosis of Some studies have also shown that sTfR is increased in blood donors, [13] during physical exercise, [14,15] cigarette smoking, [8] and high altitude, [16] where reduced oxygen tension necessitates increased erythropoiesisbut is reduced by ascorbic acid administration and iron supplementation. [17,18] A blood donor loses about 200-250 mg of iron with every unit of 450mls of blood donated. Some blood donors, especially commercial donors might donate more than three times a year for financial reasons, putting more pressure on their iron stores.
Several studies have provided reference ranges for soluble transferrin receptor concentration in the white population.
There is paucity of studies on sTfR in our environment hence this study was aimed to determine reference value of sTfR in our environment.
A study done in the United States of America showed that the reference range of sTfR receptor is 10.3 -29.1nmol/l. [12] Another study done in Kansas showed a reference value of 2.8-8.5mg/l, [19] while one done on Arabs in the Sultanate of Oman showed a reference value of 10.7-38.7nmol/l, [20] while another study showed 2.9-8.3mg/l. [21] The reference interval of serum transferrin receptor varies between different assay systems, depending on the choice of calibrators.It was proposed that recombinant serum transferrin receptor (rsTfR) preparation 07/202, may be designated a WHO Reference Reagent with assigned values of 21.7 mg/L and 30.3 nmol/L (when reconstituted with 0.50 mL distilled or deionized water). [22] Patients and Methods

Study Population:
A total of 150 participants, healthy non-fasting, males and females, within the age range of 17-60 years were selected by simple random sampling method. A research questionnaire was used to obtain basic information from participants including age, sex, tribe, occupation, level of education, history of chronic illness, nutritional history, history of smoking, menopausal status, blood donation history. Blood samples were collected afterwards for determination of full blood count and sTfR levels.
Three milliliters of venous blood was collected by venipuncture into ethylene diamine tetra-acetic acid (EDTA) bottles for each participant. Samples to be analyzed for full blood count were run immediately. Plasma samples were obtained after centrifugation of anticoagulated blood at 2500 revolutions per minute for 2 minutes. Plasma samples of subjects that met the FBC inclusion criteria and were then stored at a temperature of -70 o C. The full blood count was performed using 3 part autoanalyser (Sysmex -KX2IN ® ).
Excluded were subjects with febrile illness within 4 weeks prior to the study, pregnant and presently menstruating females, persons on iron supplements 3 months prior to study, subjects with haematological parameters outside the ranges stated above.

Correlation Between sTfR and Haematological
Parameters: There was a weak positive correlation between sTfR concentration and some haematological parameters in the study group. The r values for PCV, Hb, WBC, Platelet, and MCHC were: 0.033, 0.032, 0.093, 0.092, and 0.002 respectively but these were not statistically significant. Aweak negative correlation was observed between MCV and MCH with sTfR r= -0.042 and -0.035 respectively which were not statistically significant as shown in table 5.

Discussion
Various authors have reported varying reference values for sTfR depending on the study population. This study on apparently healthy Nigerians found the reference range of sTfR to be 0.3-3.05ug/ml. The upper limit is higher than those reported in some non-African based studies. [12,20,24] Van den Bosch G et al , [24] Allen J et al and Knox-Maculay et al reported [12,20] reference values of 0.58-2.50mg/l,0.76-2.77mg/l, 0.5-2.7mg/l respectively.
Similarly the reference range of the study population is higher than that of the Biovendor ELISA kit used for the study, which was 0.378-1.513ug/ml. [25] The mean sTfR in our study was0.89 ± 0.46ug/mL. Kohgo et al, [9] Van den Bosch G et al [24] reported lower values of 0.253±0.08ug/ ml and0.58±0.26mg/l respectively. Allen and Knox-Macaulay obtained a marginally higher mean in their studies of 1.4±0.3mg/l and 1.5±0.5mg/l respectively. Similarly, Simek M and his colleagues in another related study found mean values higher than that reported in the index study which was 1.84± 0.8mg/l. [26] Flowers and his colleagues [10] reported the sTfR value from their study to be 4.21-7.05mg/l, while Erhardt and his friends reported their own values to be 2.9-8.3mg/l. [21] These values are many folds higher than what we reported in our study, suggesting that there are variations in sTfR levels from one population to another. These variations might be due to environmental, racial, dietary and genetic factors and also assay methods used.
The sTfR levels did not differ with age.(F= 0.160, p= 0.923) Von Schmiesing et al [27] in a similar study which though found relatively higher ranges for each age group when compared to the index study, did not also observe any significant difference in the mean sTfR across the various age groups which is similar to our observations in this study. This was affirmed by Raya et al [28] and other authors. [10,11,19,24,29] This is probably because the study population, being all adults, have all attained adult levels of sTfR. Contrary to this, Jong Weon Choi et al [30] observed age related differences in sTfR levels. This difference might be due to the fact that the study participants used by Jong Weon Choi and his friends were not limited to adults alone, unlike our study and other studies. [10,11,19,24] We observed that the mean sTfR levels for both sexes were not significantly different though males had a slightly higher mean sTfR. Kohgo et al and Allen et al [9,12] in their respective study, even though the former reported lower values and the latter higher sTfR values for each sex, did not find any significant difference in their mean sTfR as also seen in other related studies. [31,32] . On the contrary Knox-macaulay and co suggested otherwise from their work, stating that there was a statistically significant difference of sTfR values in men and women. [20] Another study also noted a higher sTfR levels in females compared to males. [29] The reason for these differences cannot be readily explained, however, this might require confirmation using a larger study population. There was no significant difference in sTfR with pre and postmenopausal women (.P=0.470),suggesting that menstrual blood loss of iron does not significantly affect sTfR levels in normal adults. This observation was affirmed by Allen and his colleagues. [12] Conclusion: This study using immunological assay has established reference range forsTfR to be 0.3-3.05ug/ml in heathy adult population in Port Harcourt Nigeria.sTfR did not differ significantly across various age groups and between males and females. Menstrual status in healthy females did not significantly impact on the sTfR level.