Vitamins and Micronutrients level among Transfusion-dependent Subjects in Maiduguri, Nigeria.

Waziri Gimba, Bukar Alhaji, Obi Simon Osita, Medugu Jessy Thomus, Bukar Abdullahi Audu,
Department of Haematology, University of Maiduguri Teaching Hospital, Maiduguri
Osaro Erhabor
Department of Medical Laboratory Science, Usmanu Danfodiyo University, Sokoto.
Imoru Momodu
Department of Haematology, Aminu Kano University Teaching Hospital, Kano.
Digban Kestar Awharentomah, Jeremiah Zaccheaus Awortu.
Department of Chemical Pathology, Igbinedion University Teaching Hospital, Okada.
Olaniyan Matthew Folaranmi
Department of Medical Laboratory Science, Achievers University, Owo
All correspondence to: gimbawaziri@gmail.com.

 

ABSTRACT
Background: Blood transfusion has been very beneficial in relieving symptoms of anaemia but is associated with the risk of iron accumulation in tissues. Excess iron, by Fenton chemistry enhances oxidative stress. Antioxidant vitamins and micronutrients supplementation quenches the oxidative stress and obviate tissue damage. Objectives: The aim of the study is to evaluate some antioxidant vitamins and micronutrients level and determine whether supplementation is necessary in the management of transfusion-dependent patients. Methods: Blood samples were collected from 101 patients with ?8 units’ transfusion per year and its equivalent with no history of transfusion as controls. Serum vitamins (A and E), micronutrients (Zn and Cu), serum iron, ferritin and some haematological parameters (Haemoglobin and Haematocrit) were measured from the blood samples. Result: The underlying condition for repeated transfusion was largely SCA (96%) with male preponderance. Antioxidants, vitamins A and E were significantly decreased (p=0.000) compared to the controls, whereas micronutrients, zinc and copper levels were elevated (p>0.05 and 0.05 respectively). There was significant increase in both serum iron and ferritin in the case subjects (p 0.000). Haematocrit and haemoglobin levels was also significantly reduced in the study subjects (P<0.001). Conclusions: Our study demonstrates that the level of antioxidant vitamins was low in the transfusion-dependent subjects. Therefore, its supplementation is necessary to improve the antioxidant defence system and obviate possible tissue damage.

INTRODUCTION
Blood transfusion is an important therapeutic approach especially in patients with refractory anaemia (Khaled et al., 2013, Antoine et al., 2015). One unit of red cell transfused provides more than 200 times the normal iron intake through intestinal absorption (Crichton and Ward, 2003) and there is no major mechanism of its excretion from the body (Hoffbrand et al., 2006, Kohgoet al., 2008). Hence, in transfusion dependence, iron released from transfused red cells inevitably saturate storage sites (Salibaet al., 2015) and the unbound (free) iron molecules are available to catalysethe conversion of molecular oxygen to highly reactive oxygen species (ROS) by Fenton chemistry (Adhamet al., 2014).In addition, ROS arealso generated from the normal metabolic processes, but neutralized by the body’s antioxidant defence (Bilbiset al., 2010, Mahdi, 2014). The rate of generation of ROS and its clearance by the antioxidant defence is delicately balanced (Pamplona, 2008) and If the production exceeds the capacity of the antioxidant systems to scavenge these species, then oxidative stress ensues (McCord, 2000).Withoxidative stress resulting from antioxidant depletion, peroxidative damage to cells is evident especially in tissues that stores iron such as liver, pituitary gland, pancreas and heart (Brissotet al., 2011).Antioxidantdefence systems are intimately involved in the prevention of cellular damage (Asare et al., 2009, Lobo et al., 2010). There are several enzymes and non-enzymatic defence such as vitamins and micronutrients (Simseket al., 2005) that scavenge free radicals and greatly enhance resistance to oxidative insult (Okochi and Okpuzor, 2005). This defence is significantly diminished in oxidative stress (Dhawan et al., 2005, Rashidiet al., 2011) due to increased consumption of the antioxidants (Mainasaraet al., 2016). However, antioxidants supplementation has been reported to improve antioxidant status and precludeoxidative damage by eliminating ROS (Prasad, 2009; Arruda et al., 2013;Tukuret al., 2015).Since transfusion-dependence is prone to oxidative stress and data concerning antioxidant vitamins and micronutrients in transfusion-dependent subjects in this environment are quite limited,it is imperative to measure the exogenous non-enzymatic antioxidant status and determine whether supplementation may be necessary.

METHODS
This cross-sectionalstudy was conducted at the University of Maiduguri Teaching Hospital (UMTH) and State Specialist Hospital Maiduguri, Borno State. The ethical committees of the hospitals approved the study protocol and written informed consent was sought for and obtained from all participants. A total of 101 subjects age 20-36 years who had =8 units of red blood cells transfusions per year; and an equivalent number of age and sex unmatched normal subjects who had no history of blood transfusion were recruited for the study. Hospital records were used to determine patient’s eligibility for participation. The case subjects had history of continuous treatment with blood transfusionfor the pass at least two years.
Demographic characteristics of the patients were obtained usinga structured questionnaire. About 5 mL of blood was aseptically collected from each subject and, 3 mL and 2 mL were separately dispensed into plain and EDTA bottles respectively. The serum was harvested by centrifugation of the samples at 4000 RPM for 5 minutes and stored in cyro-vial at -20 0C until analysis.
Serum ferritin was measured using immune-enzymatic assay kit from Monobind Inc. Lake Forest, C 92630, USA while serum Iron was analysed by NITRO-PAS colorimetric method as described by Makino and colleagues (1988) using Iron Nitro-Pas mono-reagent, Centronic GmbH Am Kleinfeld 11, 85456 Wartenberg, Germany.The anticoagulated blood samples collected were tested some haematological parameters using Auto Haematology Analyser LW D3600, Shenzhen Landwind Industry Co., Ltd. Spectrophotometric methods described by Hashim and Schuttringer, (1966) and Bassey et al.,(1946) were used to analysed serum vitamin E and vitamin A respectively, while serum zinc and copper were colorimetrically estimated as described by Knoellet al. (2009) and Bartnikas, (2012) respectively using Quanti-ChromTM Assay Kits from BioAssay Systems, Corporate Place, Hayward, USA.
Statistical Package for Social Sciences (SPSS) for windows, Version 22.0 (SPSS Inc., Chicago, IL, USA) was used to express data generated as Mean ± SD. Statistical comparison of observed variables were made using student’s t-test. The level of significance (P-value) was set at less than 0.05.

RESULT
Demographic and clinical characteristics of the study subjects are presented in table 1. Demographic information showed that the age of the case subjects was not significantly different (P>0.05) compared to that of the controls. The subjects were gender unmatched but predominantly male in both case and control participants (79.2% and 56.4% respectively). The underlying clinical condition for transfusion dependence was largely sickle cell anaemia (96%). Despite the regular transfusion therapy, significant decrease in haematocrit and haemoglobin levels (20.17± 4.17 and 6.56± 1.24 respectively) was noticed in the case subjects compared to the control participants (P0.000). Although iron overload was not reported in the case subjects, serum iron indices (serum iron and ferritin) were significantly (P 0.000) elevated compared to the controls. Serum antioxidant vitamins A, E and copper were significantly lower (P0.000 and P0.05 respectively) in the case subjects compared to the controls. However, serum zinc was moderatelyincreased but there was no statistically significant difference compared with the controls (P >0.05).

DISCUSSION
Disorders with refractory anaemia are managed with repeated blood transfusion and dependence on itis associated with iron accumulation that may leads to iron overload (Cid et al., 2014). Excess iron accumulation is accompanied by increased generation of reactive oxygen species (ROS) which may outweigh body’s antioxidant defence. Disruption of pro- and anti-oxidant balance is inevitably followed by oxidative stress (Simseket al., 2005;Vaculinet al., (2010)). Non-enzymatic antioxidant supplementation has been found to be beneficial in the prevention of oxidative stress (Rashidiet al., 2011).
The present study revealed that the serum iron indices (serum iron and ferritin)were significantly elevated(P <0.05)compared to the controls, which is consistent with several other studies (Rahu et al., 2008;Simseket al., 2005;Attiaet al, 2011). Higher ferritin content was directly linked to the accumulation of iron in the tissues of the patients. However, iron overload was not observed in the case subjects largely due to the low transfusion rate. This finding is inconsistent with Cid et al., (2014);Jokhioet al., (2009) and Gao et al.,(2014)who showed many patients with iron overloaded due to much higher transfusion rate.

The result of the current study indicate that the subjects were significantly deficient of serum antioxidant vitamins A and E.Similar results have been described in other studies by Tesoriereet al., (2001); Das et al.,(2004); Dhawan et al.,(2005) in patients with thalassemia and other transfusion-dependent entities. Ironically, non-transfused (non-iron overloaded) patients with SCD have also demonstrated low levels vitamins A and E due to chronic inflammation (Buisonet al., 2004).Similarly, Arruda et al.,(2013) reported deficiency of vitamin E in70% of non-transfusion dependent SCA patients. There was no iron overload in the present study that could promote iron-induced oxidative stress that consumes the antioxidant vitamins. However, given that 96% of the case subjects were SCA patients, the vitamin deficiency observed could be pursuant to increased pro-oxidant production in the sickle red blood cells (Titus et al., 2004).
In addition to the vitamins, abundant evidence demonstrates the role of micronutrients in protecting against free radical-induced oxidative damage (Rostanet al., 2002). Although Zincdeficiency is not relatively rare in patients with SCD (Prasad, 2002), our findings revealed that serum zinc is elevated but not significant (P>0.05) compared to the controls. Similarly, Mansi et al., (2009); Rashidiet al.,(2011);Mehdizadehet al., (2008) documented increased zinc level in transfusion-dependent thalassemia patients. Regular blood transfusion which predisposes to iron overload has been suggested to increase zinc level and prevent the zinc deficiency(Alexander et al., 2000). However, the subjects in the current study were regularly transfused but there was no iron overload noticed. Nile et al.,(2008) documented that zinc deficiency induces iron accumulation or conversely increase in zinc level lessen iron accumulation. Therefore, elevated zinc level observed in the current study might have been induced by decreased iron accumulation (absence of iron overload) orabnormal glomerular filtration of zinc as seen in chronic haemolysis (Mansi et al., 2009), obvious symptom of most underlying clinical entities of the present study. This phenomenon can be exploited in transfusion-dependent subjects to prevent iron overload and its attendant consequences by maintaining normal zinc level. Copper level was significantly high (P0.05)compared the control group and this agrees with some studies (Irshaidet al., 2009; Malakaret al., 2014). Copper has an antioxidant property (Shaziaet al., 2012) and its deficiency is quite rare (Turnlundet al., 1998).
This study revealed that haemoglobin and haematocrit were significantly (p<0.05) lower compared to the controls and is in accordance with the outcome of studies by Mansi et al., (2009)and Karim et al., (2016).Anaemia is the hallmark of transfusion dependency and is reported even in non-chronically transfused subjects with SCD (Fung et al., 2007).

Conclusion and Recommendations
Our findings indicated that there was no iron overload that may induce oxidative stress which could consume the antioxidants vitamins. However, the antioxidant vitamins were deficient. Therefore,administration of antioxidants vitamins may help strengthen the antioxidant defencesystem and obviate possible tissue damagearising from pro-oxidants.Further study is suggested on the status of free radicals and endogenous antioxidants to determine whether the deficiency is coupled with enhanced free radicals’ generation and reduced endogenous antioxidants.

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Prevalence of Transfussion Transmissible Infection among Blood Donors Attending Hajiya Gambo Sawaba General Hospital Zaria

Akpulu S.P
Department of Human Anatomy,Ahmadu Bello University, Zaria, Kaduna State, Nigeria..
Adamu M
Elite International Academy of Health Technology, MTD Zaria, Kaduna State, Nigeria
Oledinma S
Dept. of Hematology, Sch of Med. Lab. Sciences, A.B.U Teaching Hospital Zaria
Yaro E.G
Department of Human Anatomy, Ahmadu Bello University, Zaria, Nigeria
Ajayi I.T
Laboratory Department, Federal Medical Centre,Birnin kudu Jigawa State Nigeria

All Correspondent to: petosw2000@yahoo.com

ABSTRACT
Background: Transfusion transmitted infections threatened the safety of patients requiring blood transfusion, which in turn imposes serious challenges for the availability of safe blood products. The aim of the study is to determine the prevalence of transfusion transmitted infections in blood donors and to evaluate the demographic characteristics of reactive and non-reactive blood donors. Materials and Methods: A retrospective study of transfusion screening tests done in Hajiya Gambo Sawaba General Hospital Zaria between January and December 2016. Data retrieved from the laboratory record books, were analyzed using simple percentages. Results: A total of 1875 blood donors with mean age of 28 was recorded. 1850(98.6%) were males and 25(1.4%) females. Three hundred and fifty nine (19.1%) donations were reactive to all screening assay. The prevalence of Hepatitis B, Hepatitis C, Human Immuno Deficiency Virus and Syphillis was found to be 127(6.8%), 190 (10.1%), 33(1.8%) and 9(0.4%) respectively. Conclusion: This study revealed that there is a substantial percentage of the blood donors harbored transfusion transmitted infections. There is a need for stringent selection of blood donors with the emphasis on comprehensive screening of donors blood using standard methods to ensure the safety of blood recipient.

Key words: Blood, donors, Screening, Transfusion Transmissible Infection.

INTRODUCTION

Transfusion-transmitted infections are increasingly becoming major mode of transmission of various diseases, in the high prevalence areas in sub-sahara Africa. This is because of high level of occurrence of blood demanding health conditions. Transfusion transmitted infections are major concern among health workers (WHO, 2008). Patients on transfusion therapy are at increased risk of transfusion transmitted infections and every year more than 90million units of blood are collected worldwide. Each transfusion carries a risk of transmitting blood-borne pathogens including mainly HIV, HBV, HCV and syphilis (Bihl et al, 2007). To improved blood transfusion safety WHO recommends an integrated strategy including establishment of well-organized blood transfusion services, prioritization of blood donation from non-remunerated donors, screening of donated blood for at least the four-major transfusion-transmissible infections (TTIs) with quality-assured assay, rational use of blood implementation of effective quality control system (WHO 2008).
Selection of donors with low TTIs risk followed by effective laboratory screening has been part of a critical process of reducing the risk of transmission to a very low level in the past 20 years (WHO, 2002). Nevertheless, particularly in low resource countries, a significant proportion of donated blood remains unsafe as it is either not screened for all major TTIs or not in a quality controlled manner, according Dodd 2007.  Most blood banks in Africa are small hospital based and relying on an important proportion of replacement donors in contrast with western transfusion unit organized with large pools of voluntary donors Dodd (2007). In addition, recommended screening tests like immunoassays (EIA) or nucleic acid testing (NAT) are technically, logically and financially still far beyond reach of many resources constrained blood banks. Every blood banks follows screening procedures to prevent such infections but infective agents escape detection due to window period, a period where in the infective agent presence cannot be detected, though it is present in donors blood (Moor 1999). Blood bank in developed countries are doing Nucleic Acid Amplification Testing (NAT) since 1999 to screened donated blood (Pathaks 2010). The window period for for testing for HIV, HBV, HCV has been significantly reduced with NAT.
The infections of HBV and human immune deficiency virus (HIV) are a rapidly growing issue of public health concern, it has been observed that HBV and HIV co infection interferes with the natural history of HBV infection and as associated with higher HBV DNA levels (Gibson 1997, colin1999). Human immune deficiency virus(HIV) infection causes a broad spectrum disease and has a varied clinical cause, from mild, flulike symptoms to AIDs, which is life threatening and the end stage of infection (MOH Mozambique 2007). Factors that can contribute to HIV transmission through blood transfusion include the window period.
Hepatitis B surface antigen testing was introduced 1970s and its transmission was consistently reduced since then (WHO 2004). Still, 300 million are infected worldwide. HBV Surface antigen is routinely included in donor screening but it fails to detect presence of HBV during window period. Chronic carriers of HBV may have low level viremia and may not have detectable HBsAg level, so some centers have started testing antibodies against HBV core protein (Diarra, 2009). Hepatitis C virus currently affect over four million peoples in USA and therefore it is the commonest transfusion transmitted infection and main indication for liver transplantation. High risk group is constituted by those who received transfusion prior to 1991 or the ones who are drug abusers using shared needles (Laperche, 2009). Incubation period can be as long as decades and this contributes to high rates of infection. Approximately 90% of individuals infected with HCV are either asymptomatic or have only mild symptoms (Matee, 2006). Syphilis is caused by infection with treponemapallidium. It is spread primarily through sexual contact. T.pallidium can also be transmitted by vertical transmission by mother to fetus or through blood if donor is already infected (Peeling2004). Approximately 57% 0f all transfusion transmissible infections and 16% of transfusion related deaths have been associated with bacterial contamination (Wagner 2004). Blood components may be contaminated with bacteria at many stages of preparation, including blood collection, processing, probing and transfusion. Bacteria may enter into blood components from many sources; exposure to donor skin bacteria by vein puncture, contaminated bags and infected environment of blood banks or hospitals, (Wagner 2004). Parasites are common infectious agents worldwide and several protozoans have shown to be transmitted via blood transfusion (Kitchen, 2006).
Malaria is endemic in tropical and subtropical regions of Africa with up to 300 million infections and one million deaths annually (Kinde et al,2000). It can also be transmitted from an infected to her fetus or from an infected blood to the recipient. WHO accepts the use of rapid and simple serological assays for TTIs and quality controlled (WHO, 2009) and Rapid test-based screening protocols still tends to be used increasingly in African blood banks. Therefore, the need to investigate the prevalence of transfusion transmissible infections among blood donors at Hajiya Gambo Sawaba General Hospital, Zaria Nigeria.

Materials And Methods
This retrospective study was conducted at blood transfusion unit of Hajiya Gambo Sawaba General Hospital Zaria, Kaduna state. Three hundred and ninety blood donors were prospectively recruited in the study from 1 January 2016 up to December 2016. Only Physically fit 18-55years old were included in the study, donors were excluded if they were below 18 years old, weigh less than <50kg, anemic, had a history of jaundice, malaria, asthma, engage in high risk behavior (i.e unsafe intercourse, drug abuse) had a past history of HBV, HCV, HIV-1 and HIV-2 or syphilis or were apparently unhealthy or malnourished. After the prospective donor has passed the hemoglobin check. Determine and uni-gold rapid test kits were used to test for HIV. The single rapid kit used for testing Hepatitis B surface Antigen (HBsAg) is ACON while Dialspot is used for testing Hepatitis C virus (HCV) and also syphilis. Blood donation process is stopped when any of the donors is reactive for any of the TTI markers. Data on blood donor’s age, sex, blood group, serological result of HCV, HBsAg, HIV and syphilis were collected at the time of blood collection, by using a structured questionnaire. Summary of statistics such as frequencies and percentages were computed. The results were presented using tables, charts and graphs.

The findings of this study showed that male is more than female. This is expected because traditionally, female is not encouraged to donate blood as the male. The study also shows hepatitis B and C virus, HIV and syphilis antibodies to be 10.1%, 6.8%, 1.8% and 0.4% respectively. The result of HBsAg in this study varied from that of Olaleye, et al (2007) which shows a very high prevalence (30.8%) of HBsAg in Ibadan. Other studies in Nigeria have also reported higher prevalence of 15.8% in University of Maiduguri Teaching Hospital Borno State, and 13.4% in Abakaliki by Idiona (2014), while Alikor and Erhabor (2007) reported a 12.4% prevalence among children in tertiary hospitals Niger Delta, Nigeria. However, the prevalence reported in this study is higher than the 8.3% prevalence reported in previous study conducted by Luka et al., at Ahmadu Bello University Teaching Hospital Zaria Kaunda state. It also higher than the 7.6%, 9.3% and 3.9% prevalence reported by Ugwuja et al(2009) in Nnewi, Awka and Abakalikl, Nigeria respectively.
The sero prevalence of HCV antibody found among blood donors in this study is lower when compared to the same study carried out in other locations, A study carried out in South Eastern state of Nigeria by Chukwurah, et al., (2005) found that 7.6% of blood donors had HCV revalence. In similar study carried out in Lagos, Nigeria, Afolabi, et al., (2012) reported that 8.4% of blood donors were positive for HCV antibody. This study agrees with the 6% HCV prevalence report by Egah et al.(2007), in Jos, Plateau state Nigeria.
Marked variation of HIV antibody has been reported in different part of Nigeria. Some workers reported the prevalence of HIV antibody to be lower than 1.8% as found in this study, while others reported higher prevalence of HIV antibody in other studies. Elele, et al., (2013) reported 4% prevalence of HIV antibody among blood donors in Port Harcourt, South Southern Nigeria had. Over all, the prevalence of HBsAg, HCV, HIV and syphilis antibodies found in this study is among the lowest reported in Nigeria. This observation may portably be due to the positive impact of HIV education and public enlightenment which have been in place in Zaria for many years. This might have helped in the reduction of the spread of HIV, HBsAg, HCV and syphilis since they have similar modes of transmission. Another factor that may account for the low prevalence of transfusion Transmissible infections in this study may be due to the fact that most of the donors were not commercial donors.

Conclusion
Results of the study which showed that the prevalence of HBsAg, HCV, HIV and syphilis antibody were 10.1%, 6.8%, 1.8% and 0.4% which suggest that HBV and HCV infections is still a health challenge that must be given more attention.

Acknowledgment
We are grateful to the Management of Gambo Sawaba General Hospital Zaria especially the Head and staff of the Laoratory for permission and cooperation during the work. We are also grateful to the staff of Elite International Health Academy Zaria for support and assistant.

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