A Publication of the Haematology and Transfusion Science Society of Nigeria.

African Journal of Laboratory Haematology and Transfusion Science
A Publication of the Haematology and Transfusion Science Society of Nigeria.
Volume 1, Number 1, May, 2022
ISSN: 2814-0591 (Print)
ISSN: 2814-0605 (Online)
Prof Teddy Charles Adias
Federal University Otueke, Bayelsa State, Nigeria
General Haematology Section
Dr. Nancy Ibeh
Nnamdi Azikiwe University, Awka, Nigeria
Thrombosis and Haemostasis Section
Prof. Ifedayo O. Ajayi
University of Benin, Benin City, Nigeria
Transfusion Science Section
Prof. Josephine Akpotuzor
University of Calabar, Calabar Nigeria
Prof. Zaccheaus. A Jeremiah, Rivers State University, Port Harcourt, Nigeria
Prof. Osaro Erhabor, Usmanu Danfodiyo University, Sokoto, Nigeria
Dr M A Muhibi,  Edo State University, Uzairue, Edo State, Nigeria
Dr Marcus Chilaka,  University of Bradford, West Yorkshire, United Kingdom
Dr Tosan Erhabor, Medical Laboratory Science Council of Nigeria, Abuja Nigeria

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Blood Cell Count Rations in Post Operative Breast Cancer Patients on Chemotherapy


Original Article 

Blood Cell Count Ratios In PostOperative Breast Cancer Patients On Chemotherapy 

Joseph E. Udosen‘, *Euphoria C. Akwiwu?, David U. Akpotuzor’, Josephine 0. Akpotuzor? 


Department of Surgery, University of Calabar, Calabar. Department of Haematology and Blood Transfusion Science, University of Calabar, Calabar. 

Introduction: Neutrophil–to–lymphocyte ratio and platelet–to-lymphocyte ratio are thought to be better in predicting systemic inflammation and thrombotic tendencies. These risks are inherent in the treatment options such as surgery and chemotherapy which are relatively available to breast cancer patients in Nigeria. This study therefore considered the assessment of these blood cell count ratios among breast cancer patients who had undergone surgical removal of the tumour as well as those on a post–operative six–course chemotherapy. 

*Corresponding Author Dr. Euphoria C. Akwiwu Department of Haematology and Blood Transfusion Science, University of Calabar, Calabar, Cross River State Nigeria Email: ecakwiwu@gmail.com 

Methods: This study was conducted at University of Calabar Teaching Hospital in Calabar, Cross River State of Nigeria. The blood cell counts were carried out by automation. Statistical analysis of data was done using SPSS 22.0. A p–value of < 0.05 was considered to infer a statistically significant difference. 

Results: This study recorded significant decreases in the haematocrit level, haemoglobin concentration, total white blood count as well as the absolute neutrophil and lymphocyte counts of the breast cancer patients compared to control subjects. There was also a significant increase for platelet–to lymphocyte ratio among breast cancer patients compared to control subjects The progression of the chemotherapy courses resulted in an initial decline of both neutrophil–to–lymphocyte and platelet–to–lymphocyte ratios up till the third course, after which upward surges were observed. 

ReceivedMarch 06, 2022, Accepted: April 28, 2022, Published: May 10, 2022 

Conclusion: Apart from anaemia and relative leucopenia, the enrolled breast cancer patients were generally observed to be at risk of thrombosis. Risks of systemic inflammation and thrombosis appear to be higher immediately after surgery and from the fourth course of chemotherapy to the sixth. 

Key words: Breast cancer, chemotherapy, thrombosis, inflammation 

AJLHTS Volume 1, Number 1, May, 2022 | ISSN: 2814–0591 (Print) | ISSN: 2814–0605 (Online) 




Epidemiology of cancer in resource–poor settings as we have in Nigeria is fraught with the challenges of underreporting and late–stage presentation arising from lack of awareness and poor health infrastructure 12 Available data across the globe has shown that breast cancer is among the leading contributors to cancer– associated morbidity and mortality particularly among women‘s. Already health challenges with female preponderance have been reported locally ranging from relatively short–term medical cases of anaemia and malaria to conditions requiring long–term medical attention such as HIV infection and diabetes 68. These reports collectively highlight the need for more intentional health interventions in support of maternal health. While the foregoing may be applicable to conditions with long–term survival rates with proper management, cancer management remains precarious with varying disease progression and survival rates. Thus, breast cancer which is predominantly seen in women, although it affects a lower proportion of males, constitutes a significant area of interest in maternal healthcare as cancer–related mortality exceeds the combined mortality from HIV infection, tuberculosis and malaria‘. 

Currently, medical management for breast cancer in Nigeria depends on certain factors such as disease staging, availability of treatment and of course patient‘s preference among other factors but generally includes in an increasing order such options as radiotherapy, surgery and chemotherapy ?. Apart from the contributions of laboratory analyses to disease diagnosis, laboratory tests are important in the monitoring of treatment. Estimation of blood cell counts are commonly utilized for this purpose to essentially aid review anaemia, inflammation and the risk of bleeding. In this 

regard, direct assessment of blood cell counts report for the level of haematocrit, concentration of haemoglobin, total and absolute differential white cell count as well as platelet count were traditionally considered to be enough. In recent times though, deriving ratios from some of the full blood count components is gaining popularity for better management of disease conditions. Notably, neutrophil–to–lymphocyte ratio and platelet to–lymphocyte ratio have been reported to be important morbidity indicators compared to the individual parameters 10.11. They are thought to be better in predicting systemic inflammation and thrombotic tendencies. Such information would be helpful for better management of breast cancer patients as both surgery and chemotherapy carry some risk of adversely affecting blood vessels and promoting thrombosis. This study therefore considered the assessment of these blood cell count ratios among female breast cancer patients who had undergone surgical removal of the tumour as well as those on a post–operative six–course chemotherapy. 

Materials and methods This study was conducted among thirty–five post–operative breast cancer patients who were accessing healthcare at the University of Calabar Teaching Hospital in Calabar, Cross River State of Nigeria. They were all females aged between 30–50 years. An equal number of age–matched apparently healthy non–pregnant women who were not on any form of medication as at the time of the study served as controls. This study employed purposive sampling technique in enrolling the post operative breast cancer patients at different courses of chemotherapy. There were 7 categories comprising pre–chemotherapy, first course through to the sixth course. An equal 

AJLHTS Volume 1, Number 1, May, 2022 | ISSN: 2814–0591 (Print) | ISSN: 2814–0605 (Online) 


Table 1.Blood cell parameters of Breast Cancer Patients and Control subjects 


Breast cancer 






HCT (1/1) 

0.31 + 0.02 

0.37 + 0.02 


Hb (g/1) 

98.74 + 6.34 

120.71 + 6.65 


WBC (x 10°/1

4.61 ± 1.26 

5.94 + 1.50 


NEUT (x 109/1

2.18 + 1.01 

2.89 + 1.01 


LYMPH (x 10°/1) 

2.34 1.00 

2.95 + 0.86 


MONO (x 10°/1) 

0.07 0.01 

0.07 + 0.01 


EOSINO (x 10°/1) 

0.02 + 0.01 

0.02 0.01 


PLT (x 10°/1) 

210.80 + 38.29 

219.14 + 56.23 



1.19 + 0.92 

1.03 + 0.38 



112.75 + 78.99 

78.69 + 26.62 


Values are expressed as mean I standard deviation, HCT = Haematocrit, Hb = Haemoglobin concentration, WBC = White Blood Cell Count, NEUT = Absolute Neutrophil count, LYMPH = Absolute Lymphocyte count, MONO = Absolute Monocyte count, EOSINO = Absolute Eosinophil count, PLT = Platelet count, NLR = Neutrophil–to–Lymphocyte Ratio, PLR = Platelet–to–Lymphocyte Ratio 


AJLHTS Volume 1, Number 1, May, 2022 | ISSN: 2814–0591 (Print) | ISSN: 2814–0605 (Online) 


Figure 1. Impact of treatment progression on neutrophil–to–lymphocyte ratio among 

Breast Cancer Patients 











1ST 2ND 3RD 4TH 5TH 


Figure 2. Impact of treatment progression on platelet–to–lymphocyte 

ratio among Breast Cancer Patients 











1ST 2ND 3RD 4TH 5TH 


AJLHTS Volume 1, Number 1, May, 2022 | ISSN: 2814–0591 (Print) | ISSN: 2814–0605 (Online) 



initial decline of the mean ratio was observed up till the third course, after which the study recorded upward changes (Figures 1 and 2). 

number of 5 participants were enrolled for each of the 7 categories. Ethical approval was duly sought and obtained from The Ethics and Health Research Committee of University of Calabar Teaching Hospital. Informed consent was obtained from each study participant. 

The blood cell counts were carried out by automation using SMART–1 Hematology Analyzer from Kinghawk Technology Co., Ltd, China. This analyser was controlled and calibrated according to manufacturer‘s instructions to ensure its fitness for use. A structured questionnaire was administered by three trained interviewers to obtain biodata and socio–demographic characteristics. Statistical analysis of data (Student t–test) was done using SPSS 22.0. Results are presented as mean and standard deviation. A p–value of < 0.05 was considered to infer a statistically significant difference. 

Results The study participants comprised 70 non– pregnant women between the ages of 30 and 50 years. Half of this number (35) were post– operative breast cancer patients at different courses of chemotherapy, while the remaining 35 control subjects were apparently healthy and not on medication as the time of the study. There were significant decreases in the haematocrit level, haemoglobin concentration, total white blood count as well as the absolute neutrophil and lymphocyte counts of the breast cancer patients compared to control subjects. Additionally, a significant increase was observed for platelet–to–lymphocyte ratio among breast cancer patients compared to control subjects (Table 1). The progression of the chemotherapy courses showed some observable effect on both the neutrophil–to–lymphocyte ratio and platelet– to–lymphocyte ratio. In both instances, an 

Discussion The present study compared haematocrit, haemoglobin concentration, total white blood cell count, absolute differential white blood cell counts, platelet count, neutrophil–to lymphocyte ratio and platelet–to–lymphocyte ratio between post–operative breast cancer patients on chemotherapy and control subjects. The findings of this study are part of the initial observations in an ongoing follow up study among breast cancer patients in our locality. Significant decreases were recorded for haematocrit level and haemoglobin concentration among the breast cancer patients compared to control subjects. In fact, the recorded mean values reflected anaemic status for these subjects. Tumour–associated anaemia arises from diverse mechanisms related to both the presence of the tumour and attempts at treatment 12,1). In the case of the former, excessive nutritional demand for blood supply to the growing tumour, marrow infiltration and immune–mediated down–regulation of erythropoietic signals from the tumour play important roles in the occurrence of anaemia. In addition, the adverse effect of cancer drugs on cell populations account more for anaemia in cancer during chemotherapy. Anaemia thus, remains one of the challenges in the management of cancer. 

Concerning the advers 

ct of chemotherapy on blood cell populations, reduction in general leucocyte population and some sub–populations were seen in this study. Cancer–related leucocyte depletion heightens the risk of infection for affected subjects and often necessitates prophylactic interventions for proper management of patients. It was 





AJLHTS Volume 1, Number 1, May, 2022 | ISSN: 2814–0591 (Print) | ISSN: 2814–0605 (Online) 



interesting to observe that although no their adverse changes to vascular membrane“.IS. significant changes occurred for platelet count I nflammatory processes can arise in between both groups, a significant increase was thrombosis due to the endothelial injuries and observed for platelet–to–lymphocyte ratio thrombogenesis, while systemic inflammation among breast cancer patients compared to also modifies the coagulation process and control subjects. Platelet–to–lymphocyte ratio promotes thrombogenesis. Apparently, the has been noted to reflect the risk of thrombosis occurrence of one instigates the involvement of than direct assessment of platelet counts alone the other in a continuous vicious cycle baring and is in fact associated with poor prognosis in timely and proper intervention “9–21. The present breast cancer 15,16. This finding, thus, suggests an study observed these risks to be high existing thrombotic risk that would otherwise immediately after surgery and from the fourth go unnoticed among the patients 

course of chemotherapy to the sixth. This This research investigation also assessed finding reveals possible critical periods for close changes in neutrophil–to–lymphocyte and monitoring and management of thrombosis in platelet–to–lymphocyte ratios among breast caring of breast cancer patients. cancer patients at varying stages of post– In conclusion, the present study operative chemotherapy. Starting from post– observed anaemia and lower values of total operative pre–chemotherapy group through six white cell count, absolute neutrophil and courses of the drug administration, this study lymphocyte counts among the enrolled breast enrolled 5 participants for each of the 7 groups cancer patients. The subjects were also observed culminating into a 35–member study to be at risk of thrombosis as seen in the population. The progression of the significant increase of platelet–to–lymphocyte chemotherapy courses showed some ratio. Risks of systemic inflammation and observable effect on both the neutrophil–to– thrombosis appear to be high immediately after lymphocyte ratio and platelet–to–lymphocyte surgery and from the fourth course of ratio. In both instances, an initial decline of the chemotherapy to the sixth. mean ratio was observed up till the third course, after which the study recorded upward changes. Conflict of Interest: Authors declare no conflict The interventions of surgery and chemotherapy of interest. both increase the risk of thrombosis through 


  1. 2. 

Fatiregun OA., Bakare O, Ayeni S, et al. 10–Year Mortality Pattern Among Cancer Patients in Lagos State University Teaching Hospital, Ikeja, Lagos. Frontiers in Oncology. 20201 https://doi.org/10.3389/fonc. 2020.573036. 

Akpotuzor JO, Akwiwu EC, Okpokam DC, Keunmoe P. Analyses of haematological malignancies records from University of Calabar Teaching Hospital Calabar, 4. Nigeria (1983–2008). InternationaJournaof Natural and Applied Sciences2011;7(1): 133–6. World Health Organization. 

Fact sheets on breast cancer. https://www.who.int/news room / fact – sheets/detail/breast–cancer. Accessed March 1, 2022. Azubuike SA, Muirhead C, Hayes L, McNally L. Rising global burden of breast cancer: the case of sub Saharan Africa (with emphasis on Nigeria) and 

  1. 3. 

AJLHTS Volume 1, Number 1, May, 2022 | ISSN: 2814–0591 (Print) | ISSN: 2814–0605 (Online) 




interesting to observe that although no significant changes occurred for platelet count between both groups, a significant increase was observed for platelet–to–lymphocyte ratio among breast cancer patients compared to control subjects. Platelet–to–lymphocyte ratio has been noted to reflect the risk of thrombosis than direct assessment of platelet counts alone and is in fact associated with poor prognosis in breast cancer 15:16. This finding, thus, suggests an existing thrombotic risk that would otherwise go unnoticed among the patients 

This research investigation also assessed changes in neutrophil–to–lymphocyte and platelet–to–lymphocyte ratios among breast cancer patients at varying stages of post– operative chemotherapy. Starting from post– operative pre–chemotherapy group through six courses of the drug administration, this study enrolled 5 participants for each of the 7 groups culminating into a 35–member study population. The progression of the chemotherapy courses showed some observable effect on both the neutrophil–to– lymphocyte ratio and platelet–to–lymphocyte ratio. In both instances, an initial decline of the mean ratio was observed up till the third course, after which the study recorded upward changes. The interventions of surgery and chemotherapy both increase the risk of thrombosis through 

their adverse changes to vascular membrane “.18. 

Inflammatory processes can arise in thrombosis due to the endothelial injuries and thrombogenesis, while systemic inflammation also modifies the coagulation process and promotes thrombogenesis. Apparently, the occurrence of one instigates the involvement of the other in a continuous vicious cycle baring timely and proper intervention “9–21. The present study observed these risks to be high immediately after surgery and from the fourth course of chemotherapy to the sixth. This finding reveals possible critical periods for close monitoring and management of thrombosis in caring of breast cancer patients. 

In conclusion, the present study observed anaemia and lower values of total white cell count, absolute neutrophil and lymphocyte counts among the enrolled breast cancer patients. The subjects were also observed to be at risk of thrombosis as seen in the significant increase of platelet–to–lymphocyte ratio. Risks of systemic inflammation and thrombosis appear to be high immediately after surgery and from the fourth course of chemotherapy to the sixth. 

Conflict of Interest: Authors declare no conflict of interest. 


  1. 2. 

Fatiregun OA., Bakare O, Ayeni S, et al. 10–Year Mortality Pattern Among Cancer Patients in Lagos State University Teaching Hospital, Ikeja, Lagos. 

Frontiers in Oncology. 20201 https://doi.org/10.3389/fo nc.2020.573036. Akpotuzor JO, Akwiwu EC, Okpoka m DC, Keunmoe P. Analyses of ha e m a tological 3. malignancies records from 

University of Calabar Teaching Hospital Calabar, Nigeria (1983–2008). International Journal oNatural and ApplieSciences2011;7(1): 133–6. World Health Organization. Fact sheets 


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Some Haemostatic indices and Body Mass Index of Apparently Healthy Deskbound and non-Deskbound Employees of a Tertiary Institution in Port Harcourt, Nigeria.


Original Article:

Some Haemostatic Indices and Body Mass Index of Apparently Healthy Deskbound and Non-Deskbound Employees of a Tertiary Institution in Port Harcourt, Nigeria

*Evelyn Mgbeoma Eze., Beauty Eruchi Echonwere-Uwikor and, Bera Hachikaru Wechie

Department of Medical Laboratory Science, Rivers State University, Port Harcourt, Nigeria


*Corresponding author:

Evelyn Mgbeoma Eze

Department of Medical Laboratory Science,

Rivers State University, Port Harcourt, Nigeria

Email:  evelyn.eze@ust.edu.ng 


Received: March 31, 2022, Accepted: April 22, 2022, Published: May 10, 2022



Background: Prothrombin time (PT) and international normalized ratio (INR) is typically used in conjunction with activated partial thromboplastin time (APTT) to evaluate the overall integrity of the intrinsic, extrinsic and common coagulation pathway and to monitor the treatment of coagulation disorders while BMI screens for weight categories ;an important health index. A high BMI can indicate high body fatness while a low BMI connotes risks of being underweight. These extremes have obvious health challenges. The aim of the study is to determine some haemostatic indices and body mass index in apparently healthy deskbound and non-deskbound employees of the Rivers State University, Port Harcourt.

Methods: A total of hundred (100) employees of the Rivers State University were recruited for this study comprising of 37 females and 63 males, out of which 53 were non-deskbound and 47 deskbound. The deskbound employees were those that work for an average of seven hours sitting down at a stretch while the non-deskbound work for an average of less than seven hours sitting down at a stretch. Using a structured questionnaire, demographic information was collected from each participant. The weight and blood pressure (BP) of each participant was taken using a weighing scale and a MOTECH digital BP monitor respectively.  A standard venipuncture technique was used to collect 3ml of blood into 3.2% tri-sodium citrate anticoagulant (at the ratio of 9:1 blood-anticoagulant) for analysis. Activated partial thromboplastin time (APTT) and prothrombin time (PT) were analyzed using the Helena C4 semi-automated coagulation analyzer, photo-optical coagulation analyzer and the Agape test kit manufactured by Agape diagnostics Switzerland. International normalized ratio (INR) was calculated from the PT results. The data obtained was analyzed using Graphpad Prism software version 6.00 produced by Graphpad software Inc. USA. Data was presented as means, median, range and standard deviation. Comparison between two means was done using student t-test analysis.

Results: The mean ± SD of the PT, INR, APTT and BMI were 13.21 ± 1.97s, 1.03 ± 0.15, 29.7 ± 4.90s and 27.02 ± 2.95s in the same order and the reference ranges established for both deskbound and non-deskbound employees as the following values: PT (s): 9.27-17.15, APTT (s): 19.9-39.5 and INR: 0.73-1.33. Comparison of the prothrombin time (PT), international normalized ratio (INR), activated thromboplastin time (APTT) and BMI between non-deskbound and deskbound staff showed a statistical significant difference for APTT (P=0.0119) and BMI (P=0.0427) while the PT and INR showed no significant statistical difference. However, all the measured haemostatic parameters were within the reference ranges.

Conclusion: The increased APTT observed among the deskbound employees is a pointer to an abnormal haemorrhage if their sedentary working lifestyle continues without any ameliorative measures. Also the increased BMI seen among the Deskbound employees connotes overweight and its attendant health consequences. It is therefore recommended that the deskbound employees should make it a point of duty to walk around in-between their working schedules to break the long hours of sitting down.

Key words: Deskbound, Non-Deskbound, Employees, Haemostatic, Body mass index, Indices 


Haemostasis is a complex process that is contingent on the complex interaction of platelets, plasma coagulation cascades, fibrinolytic proteins, blood vasculatures and cytokine mediators [1, 2, 3]. Upon tissue injury, the haemostatic mechanism employs a plethora of vascular and extravascular receptors, in accordance with the blood components, to seal off the impairments to the vasculature and closing it off from the encircling tissues [1,2, 3].

In normal healthy individuals, the haemostatic system is really a delicate balance of pro-coagulant and anticoagulant factors. Initially on vessel injury, pro-coagulant forces dominate resulting in a fibrin/platelet clot, which seals off the injured blood vessel. Once the endothelium has healed, anticoagulant and pro-fibrinolytic factors come into play allowing clot lysis and restoring blood vessel patency [4]. When this equilibrium becomes compromised under any condition, this may lead to thrombotic or bleeding complications [4].

The haemostatic system acts to coordinate the delicate balance between bleeding and clot formation. Formation of a blood clot, or thrombus, is essential to prevent bleeding in the event of vascular injury; however, inappropriate thrombus formation can cause significant morbidity and mortality [5]. 

The prothrombin time is a measure of the integrity of the extrinsic and final common pathways of the coagulation cascade [6]. It is one of several blood tests routinely used in clinical practice to evaluate the coagulation status of patients. More specifically, PT would detect deficiencies of factors II, V, VII, and X, and low fibrinogen concentrations [7,8 ].

Prothrombin time/international normalized ratio and activated partial thromboplastin time results together can help in diagnosing various haematologic disorders [9]. During oral anticoagulant therapy most of these factors are depressed, as also during the deficiencies of clotting factor activity which may be hereditary or acquired. There must be sufficient quantity of each coagulation factor, and each must function properly, in order for normal clotting to occur. A decrease can lead to excessive bleeding; too much may lead to excessive clotting [10].

The risk of thrombosis and the consequent cardiovascular events are closely coupled to aging and lifestyle factors such as a diet, smoking, and physical inactivity. Sedentary lifestyles increase all causes of mortality, double the risk of cardiovascular diseases, diabetes, and obesity, and increase the risks of colon cancer, high blood pressure, osteoporosis, lipid disorders, depression and anxiety. A physically active lifestyle therefore protect against cardiovascular disease [11, 12, 13]

Lifestyle changes, such as regular physical activity, have protective effects on cardiovascular disease, such as acute myocardial infarction and stroke [14] and habitual physical activity is accordingly recommended for populations at risk [15, 16].  

Overweight and obesity, assessed either by body mass index (BMI), a measure of weight in kilograms divided by the square of height in meters, or waist-to-hip circumference ratio (WHR), are associated with increased cardiovascular morbidity and mortality [17,18].  Indeed, there is increasing evidence that moderate weight loss could result in regression of coronary arterial lesions and significantly reduces cardiac events and total mortality [19]. Increased body mass index correlates with increased high blood pressure, increased low-density lipoprotein, increased triglycerides, increased high blood sugar and increased inflammation. All these translates to increased risk for coronary heart disease, stroke and cardiovascular death.

Various markers of haemostasis and fibrinolysis have been identified as independent cardiovascular risk factors [20] and have been widely used in clinical practice. Standard coagulation assays include assessment of activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) [21]. The assays are functional and evaluate the rate of clot formation when the coagulation cascade has been activated. In fact, APTT and PT are plasma markers of thrombogenicity.

The relevance of assessing the PT, INR and APTT and BMI in apparently healthy individuals is critical in evaluating their risks of developing coagulation disorders including thromboembolic and bleeding disorders. There is a paucity of data on the impact of lifestyle and environment on these parameter among university deskbound and non-deskbound employees hence this study

Materials and Methods

A total of 100 apparently healthy subjects comprising of 53 non-deskbound (14 females and 39 males) and 47 non-non-deskbound (22 females and 27 males) within the age range of 24-63 years were randomly selected from ten departments (Medical Laboratory Science, Chemistry, Physics, Mathematics, Animal and Environmental Biology, Bursary, Computer, Accounts, Microbiology and Plant and Biotechnology) of the university to participate in this study. Subject equal to or greater than 65 years; those suffering from known thrombotic disorders; those with high blood pressure and diabetes and those who have had a major ill-health in the last three months were excluded from the study. The weight and height of each participant was measured in kilogram using a measuring scale. The standing height of each participant was measured to the nearest centimetres (cm) using a measurement tape while blood pressure was measured using MOTECH digital BP monitor. A well-structured questionnaire was used to obtain the demographic information of the participants.

Three millilitres (3mls) of venous blood was collected into 3.2% tri-sodium citrate anticoagulant tube as described by [22]. A ratio of 1:9 of the 3.2% tri-sodium citrate and the blood respectively were mixed in the tube gently. The samples were then centrifuged for fifteen minutes at 3000rpm to obtain citrated platelet poor plasma. The supernatant plasma was then transferred into a plain test tube immediately. The sample was analyzed within two hours of collection.

Samples for APTT and PT were analyzed using the Helena C4 semi-automated, photo-optical coagulation analyzer (Helena Laboratories, United Kingdom) and the Agappe test kit manufactured by Agappe diagnostics Switzerland (Lot number: 301101769 Expiry date: March 2023). International normalized ratio was calculated from the result of the PT. The data obtained was analysed using Graphpad Prism software version 6.0 produced by Graphpad software Inc. USA. Data was presented as median, range, mean and standard deviation and comparison between two means was done using student t-test analysis with level of significance set at P= <0.05. Results were presented in Tables.


Details of Demographics, Body Mass Index and Blood Pressure of Both Non-deskbound and Deskbound Participants 

A total of 100 apparently healthy participants aged between 24-63 years were enrolled for the study comprising of 53 non-deskbound staff (14 females and 39 males) and 47 deskbound staff (22 females and 27 males). The weight range, height range, systolic and diastolic blood pressure range were 65-102kg, 1.57-1.88m, 100-134mmHg and 70-90mmHg in the same order. The demographic details of the participants are shown in Table 1.

Table 1Details of Demographics, BMI and Blood Pressure of Both Non-deskbound and Deskbound Participants


Number / Range

Total No. of Subjects

100 (Females: 37; Male: 63)

No. of Non-Deskbound Staff

53 (Females: 14; Males: 39)

No. of  Deskbound Staff

47 (Females: 22; Males: 27)

Age Range(years)


Weight Range in Kilogram(Kg)

65 – 102

Height Range in Metres

1.57 – 1.88

Body Mass Index (BMI)

21.5 – 37.7

Systolic Blood Pressure Range (mmHg)

100 – 134

Diastolic Blood Pressure Range (mmHg)

70 – 90


Range of the Haemostatic Parameters in Both Non-deskbound and Deskbound Participants

Table 2 show shows the mean, standard deviation, median and range of PT, INR and APTT of the participants. The range of PT, INR, and APTT were 9.27-17.5s, 0.73 – 1.33 and 19.9 – 39.5s in the same order.

Table 2: Range of the Haemostatic Parameters in Both Non-deskbound and Deskbound Participants


Mean ± SD


Range (Min-Max)

PT (seconds)

13.21 ± 1.97


9.27 – 17.15

APTT (seconds)

29.7 ± 4.90


19.9 – 39.5


1.03 ± 0.15


0.73 – 1.33

 Key: PT- Prothrombin time; APTT- Activated partial thromboplastin time; INR- International normalized ratio; BMI- Body mass index.

Comparison of Mean ± Standard Deviation of the Studied Parameters between Non-deskbound and Deskbound Employees

Comparison of the PT, INR, APTT and BMI between Non-deskbound and deskbound employees showed a statistical significant increase for APTT (P=0.0119) and BMI (P=0.0427). The PT and INR showed no statistically significant difference as shown in Table 3.

Table 3Comparison of Mean ± Standard Deviation of the Studied Parameters between Non-deskbound and Deskbound Employees





PT (seconds)

13.27 ± 1.82

13.15 ± 2.14

0.7685 (NS)

APTT (seconds)

28.59 ± 4.24

31.04 ± 5.30

0.0119 (S)


1.03 ± 0.14

1.02 ± 0.16

0.7214 (NS)


26.42 ± 3.08

27.84 ± 3.80

0.0427 (S)

 Key: PT- Prothrombin time; APTT- Activated partial thromboplastin time; INR- International normalized ratio; BMI- Body mass index; S – Significance; NS – Non-significance.


Prothrombin time and activated partial thromboplastin time are primary screening tests for coagulopathies [23]. In this study, the reference range obtained for PT (seconds) was 9.27-17.15, APTT (seconds): 19.9-39.5 and INR: 0.73-1.33 were within the normal range for PT (11-14 seconds) [15], 25-37 for APTT and a value below 1.1 for INR [24]. This is not in tandem with the upper and the lower limits of the study by Ihua et al. [25] which showed a range of 13.68-16.22s, 1.04-1.1 and 32.08-36.92s in the same order for apparently healthy controls recruited for a study conducted among diabetic patients in Port Harcourt but agrees with a study by Nnenna et al. [26] who obtained mean PT and APTT values of 13.60 and 32.56 respectively for apparently healthy controls in a study conducted in Calabar, Nigeria. The findings in the study is also consistent with a study by Buseri et al. [27], who reported a mean PT value of 12.9 seconds and that Seyoum et al. [28] conducted in Ethiopia which revealed a mean PT value of 13.6s, APTT value of 28.0s and an INR value of 1.16 for the apparently healthy controls used in their studies. The findings in this study is also consistent with the mean PT of 13.0s but lower than the mean APTT value of 34s found among apparently healthy males in a study conducted in India by Ahmed et al.[29]. The values for PT and APTT from the study are also in agreement with a study by Jiskani et al. [23], which revealed a mean PT, INR and APTT value of 12.56s, 0.9 and 31.49s in that order in apparently healthy control subjects in a study for hypertensive patients.

Comparison between the PT, APTT, INR and BMI of non-deskbound and deskbound employees revealed no statistical difference for PT and INR. However, the APTT and BMI was statistically significant with an increase in the APTT and BMI in the deskbound employees. The variation in BMI and APTT could be as a result of the different number of sedentary working hours as required by the nature of their job descriptions.


In this study, the prothrombin (PT), international normalized ratio (INR) and activated partial thromboplastin time (APTT) among apparently healthy non-deskbound and deskbound employees of the Rivers State University were within the normal ranges even though APTT was significantly higher in deskbound than non-deskbound employees. Increased or prolonged APTT is a pointer to an abnormal haemorrhage. Increased BMI seen among the Deskbound employees connotes overweight and its attendant health implications.


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Some Haematological Parameters of Fuel Pump Attendants in Calabar Metropolis


Original Article:


Some Haematological Parameters of Fuel Pump Attendants in Calabar Metropolis


Joyce Ezekiel Etura1, Oluchi Deborah Onwukwe1, Enosakhare Aiyudubie Asemota1, Uwem Okon Akpan 2*, Anthony Ogbonnaya Emeribe1

1Department of Haematology and Blood Transfusion Science, Faculty of Medical Laboratory Science, University of Calabar, Calabar, Nigeria.

2Department of Clinical Chemistry and Immunology, Faculty of Medical Laboratory Science, University of Calabar, Calabar, Nigeria.


*Corresponding Author

Mr Uwem Okon Akpan

Department of Clinical Chemistry and Immunology,

Faculty of Medical Laboratory Science, University of Calabar,

PMB 1115, Calabar, Nigeria. Email/Tel: akpauwem11@gmail.com/ +2347031632967


Received: March 03, 2022, Accepted: April 08, 2022, Published: May 10, 2022





Introduction: Occupational exposure to petroleum products and fumes has been reported to have toxic effects on various organs and body system including the circulatory system. This study examined some haematological parameters of the fuel pump attendants in Calabar Metropolis.

Methods: One hundred (100) male and female participants (18-50 years) were recruited into this case control study. They consisted of 50 fuel pump attendants (28 males and 22 females) and 50 controls, all residing in Calabar metropolis. The haematological parameters were analyzed using the Sysmex XE- 2100 haematology autoanalyser.  Data was analyzed using SPSS version 21.0 with Student’s t-test and Pearson’s correlation. P <0.05 were used considered statistically significant.

Results: Ninety percent of the fuel pump attendants were exposed for duration of 1-4 years. The packed cell volume (PCV), haemoglobin, mean cell volume (MCV) and neutrophil count of fuel pump attendants were significantly lower compared to the controls. Monocyte count was significantly higher in the fuel pump attendants than in the controls (p<0.05). Platelet count correlated positively with TWBC (r=0.500, p=0.000) and neutrophil count (r=0.373, p=0.008) and negatively with lymphocyte count (r=-0.337, p=0.017).

Conclusion: This finding implies that fuel has the potential to cause alterations in some haematological parameters, particularly in the red cell parameters and differential white cells, thus interfering with their normal function.

Keywords: Red cell indices, fuel pump, leucocyte count, haemoglobin



Petrol is a mixture of volatile hydrocarbon, while diesel fuel is a distillate of petroleum which contains paraffin, alkenes and aromatics1.Fuel (petrol and diesel) filling station attendants are exposed to a mixture of hydrocarbons in fuel and to the gases from vehicular exhaust2. In filling stations, the volume of fuel dispensed as well as the ambient temperature contributes significantly to the increased emission of volatile hydrocarbons. Benzene could be considered to be the most hazardous; xylene and toluene have toxicities in line with other aromatics of lower concentration compared to benzene3. Certain people have a greater risk of exposure to gasoline vapours; these include filling station workers, service station attendants, drivers of gasoline trucks and refinery workers. The volatile nature of petrol products makes them readily available in the atmosphere any time it is dispensed especially at the petrol filling station4.

Many of the harmful effects seen after exposure to gasoline are due to the individual chemicals in the gasoline mixture such as benzene, lead and oxygenates. Breathing small amount of the gasoline vapour can lead to nose and throat irritation, dizziness, headache, nausea, vomiting, confusion and breathing difficulties. Some effects of skin contact with gasoline include rashes, redness and swelling. Allergic reactions (hypersensitivity) have been reported but these are rare occurrences5.

Occupational exposure to petroleum products and fumes has been reported to have toxic effects on various organs and body system with high impact on the human respiratory system. Organs such as the heart, lung, liver, skin and kidney are affected by these toxic effects resulting in various disease and different forms of genotoxic, mutagenic, immunotoxic carcinogenic and neurotoxic manifestations 6. With the fumes from petroleum products and lead content in fuel having a negative impact on the organs of the body, there will be an impact on the haematological parameters7. Haematological parameters, including red and white blood cell counts and haemoglobin concentration are widely used as clinical indicators of health and disease. These traits are tightly regulated in healthy individuals and are under genetic control. Mutations in key genes that affect haematological parameters have important phenotypic consequences.Values outside the normal ranges are diagnostic for disorders including cancer, immune disease and cardiovascular disease7.

The adverse health effects of petrol exposure may be primarily related to impairment of the haemopoietic system with bone marrow depression8. The paucity of information on the possible effects of long term (6months and above) exposure of petrol fumes on the haematological parameters of pump attendants in Calabar metropolis locality is what informed this study.


Study area

This research study was carried out within Calabar metropolis consisting of Calabar Municipal and Calabar south Local Government Areas of Cross River State, Nigeria.

Study design/Subject selection

A case control study design was used for this study. A total of one hundred (100) subjects (males and females) aged between 18-45 years were recruited for the study. This comprised of 50 fuel pump attendants and 50 control subjects. A well-structured questionnaire was administered to obtain demographic data and their informed consent to participate in the study was obtained.

Inclusion criteria

Fuel pump attendants (exposed for one year and above) and controls who gave their consent were enrolled for the study.

Exclusion criteria

Fuel pump attendants (below one year of exposure) and controls who did not give their consent and those with blood related diseases were excluded from the study.

Ethical Approval

Ethical approval was obtained from the ethical committee of Cross-River State Ministry of Health, Calabar.

Sample collection

Using aseptic technique, 4mls of blood was collected via venepuncture into a dipotassium ethylene diamine tetra acetic acid (EDTA K2) container to a final concentration of 2mg/ml. The container was properly mixed, properly labelled and carefully placed in a rack and stored inside a cooler with ice-pack placed at the bottom of the cooler and cotton wool placed in between the ice pack and the rack containing the blood sample to avoid lyses. The anti-coagulated blood sample was used to determine the haematocrit value, haemoglobin concentration, red blood cell count, red cell indices, total white blood cell count, differential white blood cell count and platelet count.

Laboratory analyses

The haemoglobin concentration, haematocrit value, red blood cell count, red cell indices, total white blood cell count, differential white blood cell count and platelet count were analysed using System automated haematology analyser, Sysmex XE- 2100 9. Red blood cell morphology was manually evaluated by a well- trained and certified Laboratory Scientist.

Statistical analysis

Results were presented in tables as mean ± standard deviation (SD). The Statistical Package for Social Sciences (SPSS) version 21.0 was used in the statistics analysis. Student’s t-test and Pearson’s correlation were used to analyse the data. A  P-value less than 0.05 was considered statistically significant.



The demographic data obtained from the study showed that majority of the participants (74-80%) were between the ages of 18-28 years with more males (22) than females (15). Fifty four (54%) of the fuel pump attendants had no residual knowledge of the effects of petroleum products on human health (Table 1). A comparison of the haematological parameters of the fuel pump attendants and controls showed that packed cell volume (PCV), Haemoglobin (Hb) concentration, mean cell volume (MCV) and Neutrophil count were significantly lower (p<0.05) in the fuel pump attendants when compared to the control subjects. The monocyte count was significantly higher (p<0.05) in the fuel pump attendants compared to the control subjects. Other parameters were comparable (Table 2).Figure 1 shows the distribution of fuel pump attendants based on duration of exposure. Out of the 50 subjects, 90% were exposed for duration of 1 – 4years, 4% were exposed for 5 – 8years while 6% were exposed for duration of 8years and above. Figure 2 shows a significant positive correlation (r = 0.500, p=0.000) between platelet count and TWBC in the fuel pump attendants. Figure 3 shows a significant positive correlation (r = 0.373, p=0.008) between platelet count and neutrophil count in the fuel pump attendants. However, figure 4 shows a significant negative correlation (r = 0.337, p=0.017) between platelet count and lymphocyte count.


It was observed in this study that a high percentage of fuel pump attendants were young single adults within the age range of 18-25 years. This agrees with the findings of Rocha et al 10. It was also observed that the males were slightly more in number than the females, 56% and 44% respectively. This is in contrast to a similar study in Elele, Nigeria11, They reported 49% for the males and 51% for the females.  A greater percentage of the fuel pump attendants had a working duration of 1-4 years. This may be because the job is seen as temporary until they get a better job offer or offered admission into the higher institution. It was also observed that 54% of the fuel pump attendants were not aware of the effects of petrol on their health due to lack of proper education and orientation.

In this study, it was observed that the packed cell volume, haemoglobin concentration, mean cell volume and neutrophil count of the fuel pump attendant were significantly reduced compared to the controls. This may be an indication of anaemia and impairment of the bone marrow haemopoetic function as a result of the effect of exposure to benzene via inhalation. Benzene is a volatile organic compound found in petroleum products including diesel and gasoline fuel 12. This observation is in agreement with the work of Ovuru and Ekweozor13.  The morphology of the red cells of the fuel pump attendants appeared as microcytic hypochromic cells and they were also crenated. This is suggestive of anaemia. White blood cells function primarily in body defense against foreign bodies and this is often achieved through leukocytosis and antibody production. The result showed a significant decrease in the neutrophil counts which is in contrast to the findings of Ita and Udofia14. This suggests leucopaenia and bone marrow toxicity which can be due to the myelotoxic action of benzene to the white blood cells.

There was a significant increase in the monocyte count of fuel pump attendants. Monocytes are involved in first line of defense and as immune regulatory cells. This significant increase may be due to the body’s response to the hazardous petrol fumes which may be perceived by the body as an infectious substance. This finding is in agreement with the findings of Emelike et al.15

The study showed a positive correlation between the platelets and the total white cells count as well as between platelet count and neutrophil count of the fuel pump attendants. This may be due to the resultant effect of the fuel fumes on the respiratory tree with resultant inflammation and allergic reactions thus leading to harmful effect on the lungs and respiratory system. This is in agreement with the findings of Tell et al.16. The study also showed a negative correlation between the platelet count and the lymphocyte count of the fuel pump attendants. This occurs when platelet releases PGH2 (a type of prostaglandin) to activate strong platelet activation, utilized by lymphocytes to form PGI2 (prostacyclin) which inhibits the platelet activation and prevent the formation of platelet plugs. This is in agreement with the findings of Wuet al.17 It occurs in cases of respiratory tree inflammation due to the inhalation of petrol fumes.


The findings from this study have shown that exposure to fuel has adverse effect on some haematological parameters particularly in the red cell parameters and differential white cells, thus interfering with their normal blood function.


We wish to acknowledge Staff and Head of Department of Haematology and Blood Transfusion Science, University of Calabar Teaching Hospital, Calabar for use of their laboratory facility.

Table 1: Demographic data of fuel pump attendants and controls


Fuel pump attendants

N (%)


N (%)


Age range (years)





18 – 28



29 – 39



40 – 50















Previous knowledge on the effect of petroleum on health












Smoking history











Drug history












NS = No Statistics.

Table 2: Some haematological parameters of fuel pump attendants and controls


Fuel pump attendants

n = 50

Control subjects

n = 50

p – value

Age (years)




RBC count (×1012/l)




PCV (l/l)




Haemoglobin conc.(g/dl)












MCH (pg)




TWBC (×109/l)




Lymphocyte (%)




Monocyte (%)




Basophil (%)




Eosinophil (%)




Neutrophil (%)




Platelet count (×109/l)




Values are expressed as mean ± SD, where: * = Significant at p < 0.05, RBC = Red Blood Cells, PCV = Packed Cell Volume, MCV = Mean Cell Volume, MCHC = Mean Cell Haemoglobin Concentration, MCH = Mean Cell Haemoglobin, TWBC = Total White Blood Cell, NS = No Statistics.


Fig.1: Distribution of fuel pump attendants based on duration of exposure









Fig. 2: Correlation plot of Platelet count against TWBC in fuel pump attendants.




Fig. 3: Correlation plot of Platelet count against neutrophil count in fuel pump attendants.




Fig. 4: Correlation plot of Platelet count against lymphocyte count in fuel pump attendants.


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Assessment of Vitamin B12, Folate levels and some Haematological parameters among females on De-Deon syrup in Port Harcourt


Original Article:


Assessment of Vitamin B12, Folate Levels and some Haematological Parameters among Females on De-Deon syrup in Port Harcourt, Nigeria

Serekara Gideon Christian1*; Ibioku Elekima1; Beatrice Wobiarueri Moore-Igwe1; Iloloba Mac-Aworu1; and Bernice Chizoba Ariolu1

1Department of Medical Laboratory Science, Rivers State University, Port Harcourt, Nigeria


*Corresponding author:

Serekara Gideon Christian

Department of Medical Laboratory Science,

Rivers State University, Port Harcourt, Nigeria



Received: February 07, 2022, Accepted: April 21, 2022, Published: May 10, 2022



Introduction: There is a disturbing trend with intake of un-prescribed doses of multivitamin syrup as a recovering medication after illness since it is believed to boost blood blood cell production. This study aims to assess the effect of De-Deon haemoglobin syrup on Vitamin B12, Folate Levels and some Haematological Parameters among Females in Port Harcourt, Nigeria. 

Methods: Ten (10) apparently healthy female subjects between the ages of 20 to 30 years were recruited for the study; blood samples were collected via standard vein-puncture technique before administration of the drug; on the fourth (4th) day, after day 3 of drug administration; and after day 6 of drug administration. Vitamin B12, and folate were analyzed using ELISA method; while haematological parameters were determined using Sysmex autoanalyzer. Data were analyzed using Statistical Package for Social Sciences computer database (Version 10.0; SPSS Inc., Chicago, IL, USA) for mean, standard deviation, analysis of variance and correlation, p-value of <0.05 was considered statistically significant.  

Results:  showed no significant change (p>0.05) in vitamin B12, folate, red blood cells (RBC), white blood cells (WBC), haemoglobin (Hb), haematocrit (HCT) and platelet count before treatment (Day 1), during treatment (day 3) and after treatment (day 6) of De-Deon syrup administration. Correlation analysis of Vitamin B12 against haematological parameters for day, 1, 3, and after day 6 showed significant negative correlation against haemoglobin level (r = 0.6731; p = 0.0373) and statistically significant positive correlation against platelets (r = 0.6731; p = 0.0165) after day 1. Statistically significant positive correlation of vitamin B12 against platelets (r = 0.6795; p = 0.0153) was observed after day 3; no significant statistically significant correlation in levels of vitamin B12 against other haematological parameters were observed. No significant correlation was also observed for folate and haematological parameters after day 1, 3, and 6.

Conclusion: The study has therefore revealed that De-Deon syrup of haemoglobin when taken within a short period of time (acute administration), showed no statistically significant effect on vitamin B12, folate and haematological parameters. However, vitamin B12 was increased albeit without any significance. Although no significance was observed in the study, the significant positive correlation between vitamin B12 and platelet count points out the relevance of vitamin B12 supplementation as it is required in the metabolism of every cell of the human body and also a cofactor in DNA synthesis in both fatty acid and amino acid metabolism.

Keywords: De-Deon’s Syrup of Haemoglobin; Vitamin B12; Folate; Haematotological Parameters


Multivitamin syrups and/or drugs are sold over the counter in medicine shops and pharmacies without prescription and are mostly abused by consumers here in Nigeria. Most persons at the slightest observation in body weight drop or immediately after recovering from illness visits the pharmacy shop and buy multivitamin syrups (blood tonic).

De-Deon of Haemoglobin Syrup is one of such blood tonics sold in Nigerian Drug Market. It is a vitamin B12 and haemolobin supplement used for correcting such conditions as; anaemia, vitamin B12 deficiencies, liver diseases, chronic fatigue syndrome, liver function, liver damage, muscle development, addisonian anaemia, strength and physical endurance, and other conditions. De-Deon Syrup of Haemoglobin vitamin B12 enriches the blood not only with haemoglobin but also with the chief amino acids which are the basic elements indispensable in the process of blood regeneration. Vitamin B12, an antipernicious factor accelerates the synthesis of proteins and favours the maturation of red globules. De-Deon of Haemoglobin Vitamin B12 Syrup contains cyanocobalamin, haemoglobin and liver extract as active ingredients. It works by carrying oxygen from the lungs to the body’s tissues; increasing the number of liver cells; normalizing the formation of red blood cells and nerve tissues. De-Deon of Haemoglobin Vitamin B12 Syrup is composed of the following active ingredients (salts); haemoglobin – 16%W/V, liver extract – 3.1%W/V and cyanocobalamin – 0.0001%W/V. [1].

The use of dietary supplement is increasing daily around the globe [2]. An estimated 30–50% increase has occurred in the rate of energy drinks and dietary supplements intake in 2010 [3]. Vitamin supplementation impact significant benefits in terms of disease prevention and treatments; and has been accepted as a measure of controlling micro nutrient deficiencies [4].

Haemoglobin is a complex ferruginous molecule whose main function is to absorb oxygen from the lungs and then distribute it to all body cells of human [5]. Lack of adequate haemoglobin in the blood is one of the major causes of anaemia [6]. Haemoglobin is the main protein found within red blood cells. It consists of two components haem and globin. Haem, an iron and porphyrin compound is 4% and globin (amino acids) is 96%. Haemoglobin’s chemical composition allows it to bind oxygen coming into the lungs for transport to the body’s tissues. From there, haemoglobin binds carbon dioxide, the main waste product of the cell’s metabolism, and releases it into the lungs, so it can be exhale out of the body. Haemoglobin gives blood its red colour [7].

Vitamin B12, generally called cyanocobalamin, is a porphyrin like ring compound with central cobalt atom attached to a nucleotide. Its anti-anaemic function has been known for years [6]. Cyanocobalamin is a manufactured form of vitamin B12 used to treat vitamin B12 deficiency [7]. A low concentration of vitamin B12 is associated with complications such as birth defects and neurological disorders [8][4][7]. More severe deficiencies of this vitamin are associated with haematological disorders such as anaemia, leukopaenia and thrombocytopaenia, promoting haematological shortages, resulting in increased mean corpuscular red cell volume (MCV) and anaemia through the alteration of erythropoiesis [9]. When haemoglobin levels falls below a certain range, an individual may develop symptoms of anaemia [6]. A deficiency in vitamin B12 can affect the production of red blood cells, decreasing the number of red blood cells circulating in the blood, and therefore decreasing the amount of haemoglobin available for oxygen transport [7]. Low vitamin B12 levels in community-dwelling adults are usually corrected with supplements [8] among which include De-Deon of Haemoglobin Syrup.

Folate is also known as folic acid, folacin, vitamin B9, Vitamin M, Folvite, Acifolic, Folcidin, and scientifically as Pteroylglutamic acid, was first found by Lucy Wills, a consultant pathologist at the Royal Free Hospital in London through her work, which resulted in correcting macrocytic anaemia of pregnancy in female textile workers in Bombay [10][11]. Folic acid was first isolated in 1941 from spinach hence its name folium (leaf) [12]. Folate is naturally present in a wide variety of foods, including vegetables (especially dark green leafy vegetables), fruits and fruit juices, nuts, beans, peas, seafood, eggs, dairy products, meat, poultry, and grains [13].

De-Deons of Haemoglobin Vitamin B12 Syrup, finds its usefulness in the treatment or management of conditions that affect red cell productions and result to anaemia. It is a very popular drug used in Nigeria and prescribed by many clinicians to their patients. Based on the wide usage of this drug on folate, it becomes imperative to determine the effects of this drug on blood production, hence the study.



2.1 Study Design

An experimental and comparative study aimed at evaluating the effects of De-Deon of Haemoglobin syrup on vitamin B12, folate and some haematological parameters among selected female subjects. The study was carried out from November, 2020 to December 2020. A total of ten (10) adult female subjects between the ages of 20 to 30 years were recruited for the study and blood samples for analysis were collected before (control), between and after taking De-Deon    of haemoglobin syrup on each interval days of collection and analyzed respectively. Samples were collected before administration of the drug, after the third (3rd) day of administration of the drug and after the sixth (6th) day of administration of the drug.

2.2 Study Area

The study was conducted in Port Harcourt metropolis, Rivers State, Nigeria. Port Harcourt, the capital of Rivers State, Nigeria.

2.3 Study Population

The subjects in this study comprised of apparently healthy adult females aged between 20-30 years. Blood samples were drawn from the ten (10) healthy volunteer subjects into tube containing 0.5ml of 1.2mg/ml dipotassium ethylene diamine tetra acetic acid (K2 EDTA) aniticoagulated-preservative container.

2.4 Inclusion Criteria

Only apparently healthy adult females were recruited for this study. Participants were eligible for inclusion in the study if they were females between the ages of 20 to 30 years, not on drugs, menstruation, and not alcoholics.

2.5 Exclusion Criteria

Participants were excluded if they had severe anaemia (Haemoglobin <8g/dL), were suffering from acute or chronic infections that could affect their haemoglobin and ferritin levels at the time of the blood sampling, were on medication or were found to be drug addicts, were diagnosed with thalassemia trait.

2.6 Informed Consent

Informed consent was obtained from all subjects who willingly participated in the study and gave their blood samples for analysis.

2.7 Sample Collection, Transportation, Processing and Preservation

Venous blood sample was collected with the use of vacutainer needle from each subject, of which 4.0 ml of blood was collected and added into individualized vacutainer tube containing 0.5ml of 1.2mg/ml dipotassium ethylene tetra-acetic acid (K2EDTA). The samples were preserved using ice pack in a thermo cool container and then transported to the haematology laboratory where they were analysed. Full blood count was carried out, and then the blood samples were centrifuged to obtain plasma. The plasma obtained was used for vitamin B12 and folate analysis using an ELISA reader capable of reading absorbance at 450nm wavelength.

2.8 Sample Analysis/Methodologies

All samples for full blood count were analysed using the automated machine (SYSMEX, manufactured by KOBE, Japan, Model No: KX-21N). All samples for vitamin B12 were analysed using Human Vitamin B12 ELISA Kit, CALBIOTECH, Inc., El Cajon, U.S.A. Lot No VBE5948: Expiry Date: 2021/02; while samples for folate were analyzed using using Human Folate ELISA Kit, CALBIOTECH, In, EI, Cajon, U. S.A. Lot number VBE598: Expiry date: 2021/01

Determination of Full Blood Count Using Sysmex KX-21N Auto-analyser, Kobe, Japan.

Procedure of Full Blood Count Using Sysmex

The procedure is such that the sample for analysis is mixed using a vortex mixer and the lid of the sample container is opened and the sample fed into the Sysmex auto-analyser via the probe. The analysis was done by the machine and the results of the analysis displayed at the read-out screen which can be printed out.

Determination of Vitamin B12 Using Human Vitamin B12 ELISA Kit, CALBIOTECH, Inc., El Cajon, U.S.A. Lot No VBE5948: Expiry Date: 2021/02.

Procedure of Vitamin B12 Estimation Using Human Vitamin B12 ELISA Kit

The EDTA blood samples were centrifuged to obtain plasma. All reagents and specimens were allowed to come to room temperature before use. Desired numbers of coated strips were placed into the holder. 50ul of extracted Vitamin B12 standards, controls and samples were dispersed into appropriate wells. 50ul of biotinylated intrinsic factor reagent was dispersed into each well. The microplate was shaken gently for 20-30 seconds to mix and was incubated for 45 minutes at room temperature (25⁰C). 50ul of enzyme conjugate was added into all the wells, the microplate was gently shaken for 20-30 seconds to mix and was incubated for 30 minutes at room temperature (20-25⁰C). The contents were briskly shaken out of the wells, the wells were rinsed 3 times with 1X wash buffer, and the wells were stroked sharply on absorbent paper to remove residual water droplets. Using a multi-channel pipette, 100 ul of TMB Substrate was dispensed into each well which resulted in the development of a blue-coloured solution. The absorbance of the colour was read spectrophotometrically at 450nm wavelength.

Determination of Folate using Human ELISA Kit, CALBIOTECH, In, EI, Cajon, U. S.A. Lot number VBE598: Expiry date: 2021/01

Procedure for Folate Estimates Using Human ELISA Kit

The EDTA blood sample were centrifuged to obtain the plasma. All reagent and sample were allowed to come to room temperature for use. Desired numbers of coated strips were placed into the holders. 5ųl of extracted Folate standard, control and sample were dispensed into the appropriate wells. 50ųl of biotinylated intrinsic factor reagent was dispensed into each well. the microplate was shaken gently for 20 to 30 seconds to mix and was incubated for 30 minutes under a temperature of (25°C). 50ųl of enzyme conjugate was added into all the wells, the microplate was gently shaken for 20-30 seconds to mix and was incubated for 60 minutes at room temperature (20-25°C). The content was briskly shaken out of the wells, the wheels were rinsed 3 times with wash buffer and the wells were stroked sharply on absorbent paper to remove residual water droplets.  Using a multi- channel pipette, 10ul of TMB substrate was dispensed into each well which result in development of blue colour solution.  The absorbance of the colour was read spectophotometrically at 450nm wavelength.

2.9 Data Analysis

Data generated from this study was statistically analyzed using Statistical Package for Social Sciences computer database (Version 10.0; SPSS Inc., Chicago, IL, USA) to obtain mean, standard deviations, Pearson’s correlation, p-value and t-value using analysis of variance (ANOVA). Tukey’s multiple comparison tests was done to check for significance between groups. A p-value of <0.05 was considered statistically significant in all statistical comparison.



3.1 Demographic Characteristics of the Studied Subjects

A total of ten (10) apparently healthy female participants within the ages of 20 to 30 years were recruited for the study. Samples were collected from all ten (10) participants before administration of De-deon syrup which served as control, between administration and after administration of De-deon syrup. Samples were collected before administration of the drug, after the third (3rd) day of administration of the drug (4th day) and after the sixth (6th) day after administration of the drug (7th day). Details of the demographic characteristics of the study population are shown in Table 3.1.

3.2 Analysis of Variance of the Studied Parameters Before Day 1, After Day 3 of Treatment and After Day 6 of Treatment

Analysis of variance of treatment before (day 1 ), during (day 4) and after treatment (day 7) of full blood counts from the study as presented in Table 3.2, shows no significant change (p>0.05) in haematological parameters of red blood cells (RBC), white blood cells (WBC), haemoglobin (Hb), haematocrit (HCT), platelet count (PLT), mean cell volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), RDW-SD and RCW-CD before treatment (Day 1), during treatment (day 4) and after treatment (day 7) of De-Deon syrup administration.

3.3: Correlation Analysis of Vitamin B12 Against Full Blood Count for Day 1

Correlation Analysis of Vitamin B12 against FBC and Red Cell Indices before day 1 as reported in Table 3.3 shows a significant positive correlation in Vitamin B12, and Platelet (r= 0.6731 respectively) and a reverse significant correlation in HGB level (r= -0.5868) and non-significant negative and positive in WBC, RBC, HCT, MCV, MCH, MCHC.

3.4: Correlation Analysis of Vitamin B12 Against Full Blood Count after Day 3

Correlation Analysis of Vitamin B12 against FBC and Red Cell Indices after day 3 as reported in Table 3.4 shows a significant positive correlation in Vitamin B12, and Platelet (r = 0.6795respectively) and non-significant positive and negative correlation in the other parameters.

3.5: Correlation Analysis of Vitamin B12 Against Full Blood Count after Day 6

Correlation Analysis of Vitamin B12 against FBC and Red Cell Indices after day 6 as reported in Table 3.5 showed non-significant positive and negative correlation was observed in levels of all parameters

3.6: Correlation Analysis of Folate with Red Cell Indices

Table 3.6a-c shows the correlation between folate and the red cell indices (RBC, HBG, HCT, MCV, MCH, MCHC) before administration of drug, after day 3 and after day 6. No statistical significance was observed using Pearson correlation.

Table 3.1 Demographic Characteristics of the Studied Subjects



Total human subjects


Control (Before administration)


Day 4 (During administration)


Day 7 (After administration)


Number of females



Table 3.2: ANOVA Of Treatment Before (Day 1), After (Day 3) and After Treatment (Day 6) of the Studied Parameters


Before Day 1

After Day 3

After Day 6




WBC (103/µL)







RBC (106/µL)







HGB (g/dL)







HCT (%)







PLT (103/µL)







MCV (fl)







MCH (pg)







MCHC (g/dL)







RDW-SD (fl)







RCW-CD (%)





















KEY: NS= Not-significant; S=Significant; applicable to all Tables.

Table 3.3: Correlation Analysis of Vitamin B12 Against Haematological Parameters before Day 1

Vitamin B12


r = -0.2491

p = 0.2438


r = -0.1496

p = 0.3400


r = 0.6731

p = 0.0373


r = -0.4676

p = 0.0865


r = 0.6731

p = 0.0165


r = -0.2371

p = 0.2548


r = -0.2141

p = 0.2763


r = 0.0122

p = 0.4866


Table 3.4: Correlation Analysis of Vitamin B12 against Haematological Parameters after day 3

Vitamin B12


r = -0.2915

p = 0.2069


r = -0.1168

p = 0.3740


r = -0.3824

p = 0.1378


r = -0.3777

p = 0.1409


r = 0.6795

p = 0.0153


r = -0.1995

p = 0.2903


r = -0.1132

p = 0.3777


r = 0.2480

p = 0.2448

Table 3.5: Correlation Analysis of Vitamin B12 Against Haematological Parameters after Day 6

Vitamin B12


r = -0.1936

p = 0.2961


r = -0.1623

p = 0.3271


r = -0.4641

p = 0.0883


r = -0.3617

p = 0.1522


r = 0.4531

p = 0.0942


r = -0.1219

p = 0.3686


r = -0.1483

p = 0.3413


r = -0.0969

p = 0.3950


Table 3.6a: Correlation Analysis of Folate against Red Cell Indices for Day 0




RBC (106/µL)

HGB (g/dL)

HCT (%)

MCV (fl)

MCH (pg)

MCHC (g/dL)

r value








p value









Table 3.6b: Correlation Analysis of Folate against Red Cell Indices after Day 3




RBC (106/µL)

HGB (g/dL)

HCT (%)

MCV (fl)

MCH (pg)

MCHC (g/dL)

r value








p value









Table 3.6c: Correlation Analysis of Folate against Red Cell Indices after Day 6




RBC (106/µL)

HGB (g/dl)

HCT (%)

MCV (fl)

MCH (pg)

MCHC (g/dL)

r value








p value










The present study investigated the effects of De-Deon Syrup of Haemoglobin Vitamin B12 on vitamin B12, folate and some haematological parameters among selected female subjects. From the study, there was no significant increase (p>0.05) in all analyzed haematological parameters before treatment (Day 1), after treatment (day 3) and after treatment (day 6) with De-Deon syrup administration. This non-significance in the result is in agreement with results obtained by Smelt et al. [14] where they reported a non-significant measurable change in routine haematological parameters after treatment with vitamin B12 in older persons with either normal or vitamin B12 below the reference range 200-900pg/mL. The results are also in line with previous analyses in the Leiden 85-plus study, where no relationship was found between vitamin B12 and changes in MCV over time [15]. The results of the study are not in agreement with results obtained by Abed et al. [16] where they observed significant increase in haematological parameters of Hb, MCH and MCHC and no significant increase in MCV among children suffering from anaemia after receiving syrup containing vitamin B12 for 3 months [16], though the duration of administration in this study was 6 days. Also, Bolaman, [17] reported increased haematologic responses in both oral and intramuscular vitamin B12 supplemented for 1 month and observed improvements of cognitive function, sensory neuropathy and vibration sense but the difference between both groups were not statistically significant [17].

There are several possible explanations for the lack of effect. First, the study consisted of participants without any apparent vitamin B12 deficiency. As these participants may not have had a true tissue deficiency of vitamin B12, this may have diluted the effect of supplementation. Secondly, participants in this study were without clinical features of anaemia. Perhaps haemoglobin levels in non-anaemic patients are less likely to increase in response to vitamin B12 treatment than haemoglobin levels in anaemic patients [18]. Thirdly, a low vitamin B12 concentration alone may not be the only reason to develop anaemia, and treatment of these low levels may not be sufficient to raise haemoglobin levels especially when the drug was not taken for a long period of time as in the present study; samples were collected after 3 and 6 days of administration. The 3 and 6 days might not have been enough to elicit an effect which may cause a significant change in the studied parameters. However, vitamin B12 levels in the subject were not within the levels or cut off for deficiency as the reference range for vitamin B12 is 200-900pg/ml [14] although it differs according to population.

In this study, no significant change was observed in the levels of Vitamin B12 after day 3 and day 6 of treatment. Although there was a slight increase in the levels of vitamin B12, the increase was not enough to cause a significant change. This non significance in result is in agreement with results obtained by Smelt et al. [14].

De deon syrup contains vitamin B12 [1], and thus, it’s supplementation should cause a rise in its value as reported by Smelt et al. [14] who recorded an increase in vitamin B12 after supplementation without statistical significance. The non-significance in the result may be as a result of the short duration of supplement administration.

In this study, analysis was done before day 1, after day 3 and after day 6 of administration as opposed to higher duration of administration (more than 1 month) in other studies. This duration may not have been enough to elicit an effect which might be significant when statistically analyzed. This can also be seen in the correlation analysis of vitamin B12 before day 1 as presented on Table 4.3 where there was a positive correlation in haemoglobin and platelet concentration; after day 3 as presented on Table 4.4, there was a positive correlation in platelet concentration and on day 6 as presented on Table 4.5, showing that De-Deon of haemoglobin syrup might have some effect on haematological parameters with increased duration. The levels of vitamin B12 as reported on table 4.2 were also increasing from 1785±1500 before day 1, to 1863±1632 after 2 and to 2027±1729 after day 6. These observed increase and significant positive correlation may be as a result of the supplement administration where the vitamin B12 content of De-Deon syrup [1], having an additive effect on vitamin B12 level in the body resulting in the observed difference, but the increase was not enough to cause a significant difference when statistically analysed. Secondly, the study consisted of participants without any apparent vitamin B12 deficiency or just below the cutoff values. As these participants may not have had a true tissue deficiency of vitamin B12, this may have diluted the effect of supplementation.

In this study there was no significant on effects of the De-Deon hemoglobin syrup on folate. The results obtained shows no significant increase in folate after administration of the drug. Further analysis was carried out, correlating folate and red cell indices (Table 4.4a-c) but no significance was observed in the result.  There are several possible explanations for the lack of effect, first, some studies consisted mostly of participants without folate deficiency or just below the cut-off values. As these participants may not have had a true tissue deficiency of folate, this may have diluted the effect of supplementation. Secondly, many of the included studies mainly consisted of participants without anaemia. Perhaps haemoglobin levels in non-anaemic patients are less likely to increase in response to De Deon Haemoglobin treatment than haemoglobin levels in anaemic patients [18]. Unfortunately, the low numbers of people with anaemia refrained us from drawing definite conclusions on the effects of supplementation in an anaemic population with low folate concentrations. Third, a low folate concentration alone may not be the only reason to develop anaemia, and treatment of these low levels may not be sufficient to raise haemoglobin levels. Other genetic or environmental factors may be involved in the onset of anaemia [19]. Also, other causes such as chronic inflammation may play a role in the development of anaemia [20].

White blood cells (WBCs) are a part of the immune system that helps fight infection and defend the body against other foreign materials. The most important function of vitamin B12 is DNA synthesis, necessary for cell division, whereby it could modulate human immunity [21]. Addition of vitamin B12 in B12-deficient patients facilitates the production of T lymphocytes recruited in cellular immunity, restores an abnormally increased CD4/CD8 ratio and maintains the count of lymphocyte subgroups in the normal range [22]. Erkurt et al. [23] demonstrated that the intake of vitamin B12 in patients with anaemia causes an increase in the absolute number of CD8+ as well as a slight increase in CD4+ lymphocytes [23]. In this present study, B12 supplementation did not affect white blood cells as no significant increase was seen in white blood cell concentration. This is in agreement with results obtained by Lewicki et al. [24] in their study on the effect of vitamin B12 supplementation on white blood cells. The result of the study is not in agreement with results obtained by Erkurt et al. [23] and Tamura et al. [25]. The reason for this difference may be as a result of participants in our study consisting of apparently healthy subjects without any clinical features of pernicious anaemia as addition of vitamin B12 in B12-deficient patients facilitates the production of T lymphocytes recruited in cellular immunity, restores an abnormally increased CD4/CD8 ratio and maintains the count of lymphocyte subgroups in the normal range [25] and participants in their study consisted of subjects with anaemia.

Red blood cells contain a protein called haemoglobin, which carries oxygen from the lungs to all parts of the body. De-Deons of Haemoglobin syrup contains vitamin B12 which is a required nutrient that helps allow proper red blood cell production and function in the body.  Thus, its supplementation tends to increase the vitamin B12 concentration which was increased although not significant. Vitamin B12 has been seen to have a well-defined role in haematopoiesis as it is essential for normal maturation and development of blood cells [26]. Thus, an increase in Vitamin B12 should increases hematopoiesis which also increases the concentration of red blood cells (RBC), haemogblobin and also haematocrit values.

In this study, no significant increase was observed in red blood cell concentration, haematocrit and also levels of haemoglobin after day 3 and day 6 of administration, the result of this study is in agreement with results obtained by Smelt et al. [14]. The result is not in agreement with results obtained by Abed et al. [16]. The reason for this discrepancy may be as a result of the participants in the study without clinical features of anaemia. Perhaps red blood cell, haematocrit and haemoglobin levels in non-anaemic patients are less likely to increase in response to vitamin B12 treatment than the levels in anaemic patients [18]. A low vitamin B12 concentration alone may not be the only reason to develop anaemia, and treatment of these low levels may not be sufficient to raise red blood cell, haematocrit and haemoglobin levels [14] especially when the drug was not taken for a long period of time in the present study.

No significant difference was found in the levels of haematological indices of MCV, MCH and MCHC after day 3 and day 6 of treatment. Haematological indices also known as red blood cell indices provide information about the haemoglobin content and size of red blood cells. Abnormal values indicate the presence of anaemia and which type of anaemia it is. Deficiency of vitamin B12 has been known to cause a type of anaemia known as pernicious anaemia [27]. Pernicious anaemia, a type of anaemia caused by vitamin B12 insufficiency is thought mainly to be caused by an autoimmune process which inhibits the production of a substance in the stomach called intrinsic factor. This vitamin is needed to make red blood cells, which carry oxygen to all parts of the body. The non-significant increase in the results of this study is in agreement with results obtained by Smelt et al. [14] and Weck et al. [27]. The non-significant increase in the results of haematological indices of MCV, MCH and MCHC in this study is not in agreement with results obtained by Abed et al. [16].

Platelets, also called thrombocytes are a component of blood whose function (along with the coagulation factors) is to react to bleeding from blood vessel injury by clumping, thereby initiating a blood clot [5] (Waugh and Grant, 2007). In this study, no significant difference was observed in platelet concentration after day 3 and day 6 of administration of De-Deon of Haemoglobin syrup. However, a significant positive correlation between platelet volumes was reported after day 3 of administration. The result of this study is in agreement with results obtained by Smelt et al. [14]. The body needs vitamin B12 for tetrafolate production and methylation reactions that are required for DNA synthesis. In addition, B12 is required for the formation from homocysteine to methionine and the formation of shaped elements in the bone marrow. Therefore, it was reported that thrombocytopenia might occur as well as anaemia and leukopenia as a result of the damage of DNA synthesis due to B12 and/or folate deficiency which may lead to observed difference in platelet count upon treatment in vitamin B12 deficiency. In this study, participants consisted of only apparently healthy subjects without any clinical features of vitamin B12 deficiency thus resulting in the non-significant difference in the study.

 The red cell distribution width (RDW-SD) (fl) reflects erythrocyte anisocytosis and the extent variability of erythrocytes [28] and also related to inflammation. Activated red blood cells may have a crucial role in inflammation and it has been known that vitamin B12 deficiency which is a leading cause of pernicious anaemia increases the red cell distribution width [29] and it also potentiate inflammation via the red cell distribution width platelet ratio (RPR). In our study, no significant increase was seen in the levels of red cell distribution width after day 1, day 3 and day 6 of treatment with De deon of Haemoglobin syrup. RDW indicates heterogeneity and equivalent of anisocytosis of red blood cell. It was documented that high levels of RDW predicts elevated red blood cell destruction in iron, folate, and vitamin B12 deficiency [30][31]. Eventually, different-sized erythrocytes may be expressed and elevated RDW may result in increased RPR [31]. The result in our study is not in agreement with results obtained by Yin et al. [32]. The reason for the discrepancy is as a result of the participants in our study were apparently healthy subjects without any clinical features of pernicious anaemia as opposed to that in their study whose participants consisted of those with known vitamin B12 deficiency.

Red cell width (RCW-CD%) is a measure of the range of variation of red blood cell (RBC) volume that is reported as part of a standard complete blood count. Usually, red blood cells are a standard size of about 6–8 μm in diameter. Certain disorders, however, cause a significant variation in cell size. Higher RCW values indicate greater variation in size. Normal reference range of RCW-CD in human red blood cells is 11.5–15.4% [28]. Red cell width is often used together with mean corpuscular volume (MCV) to determine the possible causes of anaemia. It is mainly used to differentiate anaemia of mixed causes from anaemia of a single cause.


De-Deon syrup when taken within a short period of time (acute administration), there is no positive effect on haematological parameters rather vitamin B12 was increased albeit without any significance. Although no significance was observed in the study, the significant positive correlation between Vitamin B12 and platelet count points out the relevance of Vitamin B12 supplementation as it is required in the metabolism of every cell of the human body and also a cofactor in DNA synthesis in both fatty acid and amino acid metabolism, particularly important in the normal functioning of the nervous system via its role in the synthesis of myelin and in the maturation of developing red blood cells in the bone marrow.


Further studies are recommended which will include a large sample size and analysis of other variables such as methylmalonic acid and total homocysteine as these variables reflects a true tissue deficiency of vitamin B12. Such studies could reveal the impact of vitamin B12 supplementation on the concentrations of the mentioned biomarkers. Also, it is recommended that the duration of drug administration be increased as the limitations of the present study are the small sample size, the lack of analysis of other variables that reflect the tissue deficiency of vitamin B12, such as methylmalonic acid (MMA) and total homocysteine and low duration of drug administration. The marked areas in this study such as duration of administration and sample size should be looked into as it will help provide more robust bases to establish the effect of this drug in particular on its effect on vitamin B12 and haematological parameters.


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Single neucleotide polymorphisms in the V3 region of glycoprotein 120 among Human Immunodeficiency Virus positive subjects in Nigeria


Original Article:


Single nucleotide polymorphisms in the V3 region of glycoprotein 120 among Human immunodeficiency Virus positive subjects in Nigeria.

Stowe Opuada1, Kenneth Zifawei1, Teddy Charles Adias2, Zaccheaus Awortu Jeremiah3,

1Department of Medical Laboratory Science, Rivers State University, Port Harcourt, Nigeria

2Federal University Otuoke, Baylsa State, Nigeria

3Department of Medical Laboratory Science, Niger Delta University Wilberforce Island, Nigeria


Corresponding Author

Professor Zaccheaus Awortu Jeremiah

Email: zacjerry39@gmail.com

Tel: +234 803 404 5636


Received: January 31, 2022, Accepted: April 08, 2022, Published: May 10, 2022



Objectives: The Cysteine-Cysteine Receptor 5 (CCR5) molecule is the most important co-receptor for macrophage-tropic HIV-1. A 32-bp deletion in the gene encoding CCR5 (CCR5-del32) confers nearly complete resistance to HIV-1 infection in homozygotes, and slows the rate of progression to AIDS in heterozygous adults. This study aimed to determine the frequencies of chemokine receptor 5 (CCR5) genes alleles associated with V3 loop of glycoprotein (gp) 120 among HIV patients in Yenagoa, Nigeria.

Methods: Genomic DNA was extracted from whole 100 blood samples using Quick gDNA Mini Prep kit. A portion of the CCR5 gene and gp 120 regions of HIV-1 DNA were amplified from the gDNA by PCR with sequence specific primers and universal primers respetively followed by agarose gel electrophoresis. DNA sequencing of the resultant gp 120 amplicon was also carried out.

Results: The CCR5 gene was positive at 189bp. None was positive to the CCR5/Δ32 heterozygous and none was also positive to the Δ32 homozygous deletion. The V3 region of the HIV DNA was seen to be positive at 250bp. 

Conclusion: This study shows that there is Single nucleotide polymorphism (SNP) of the protein sequence in the V3 region of gp120 among positive individuals in the study population.


Keywords: Single nucleotide polymorphism, glycoprotein 120, Humman Immunodeficiency virus, Nigeria



Chemokines are groups of small cytokines or proteins that are secreted by cells that send immune signals. The name is as a result of their capacity to induce immune responses. All vertebrates have chemokines in them even some bacteria. There are four main subclasses of Chemokines. These are; CXC, CC, CX3C and XC. They are receptors found on the surface of target cells (1).

Chemokines and their receptors are expressed by a wide variety of non-haematopoietic cells, and that chemokine function extends far beyond leukocyte physiology. Even within the world of leukocytes, the connections among chemokines, their receptors, and human immunodeficiency virus (HIV) infection broadens the previously narrow focus on chemokines as mere chemoattractants. Furthermore, a proliferation of animal models has more precisely defined the functions of chemokines in vivo (2).

The attraction of leukocytes to tissues is essential for inflammation and the host response to infection. The process is controlled by chemokines, which are chemotactic cytokines. Chemokines majorly act as guide for cell migration during immune response. Other chemokines help in the development and growth of new blood vessels. During inflammation, chemokines are released from variety of cells against pathogens. Chemokines can also be activated to promote wound healing during inflammation, maintenance of homeostasis, and are involved in driving the leukocytes to the points of inflammation. This process is known as homing (3).

Basal Chemokines: The site of production of basal chemokines for homeostasis is the thymus and also the lymphoid tissues. Few examples are CXCR5, CCR9 and CCR10. They are produced in the bone marrow (4). Inflammatory ChemokineWhen injury or infection occurs, there is increased production of inflammatory chemokines which drive leukocytes to the points of infection or injury. Few examples are: CCL2 and CXCL1 (5).

Statement of the Problem

Sexual active age of an individual shifts significantly between various societies and varies seasonally. The act of sexual activity is part of human natural physiology though has its associated risks. The major risk factors include HIV transmission among many others. Human immunodeficiency virus (HIV) transmission in sub-Saharan Africa is mostly by heterosexual contact. Unprotected sexual intercourse places a substantial number of sexually active individuals at risk for sexually transmitted disease (STD) and HIV infection(6).

 HIV-1 infection has spread to all population groups in Africa and has reached epidemic proportions. The rate of progression of HIV-1 disease exhibits a remarkable variation among different individuals. Many host genetic factors are now known to affect the disease progression rates, especially polymorphisms in genes encoding chemokine receptors (7).

Justification of the Study

The adverse effect of HIV is global; However, sub-Saharan Africa bears most burden of HIV infection. The discovery of the delta 32 (CCR5Δ32) allele of CCR5 brought to the fore the importance of CCR5 in HIV infection and disease progression (8). It has been reported that persons who lack functional CCR5 (i.e. CCR5Δ32) in the homozygous state, may be completely protected from HIV infection. On the other hand, persons with both alleles of CCR5 are slow progressors if infected with HIV (9). In Nigeria, there is no report about the heterogeneous or homogeneous allele of the mutant CCR5.

However, it has been reported that this genetic mutation is found in Caucasian rather than non-Caucasian population (10). Genetic relationships among ethnic groups are not uniform across the geographical region.  Although no studies on chemokine receptor gene alleles have been reported in Yenagoa Bayelsa State, Nigeria. On this assumption, the need to determine the pattern of distribution of CCR5 in our geographical region becomes imperative. This study was therefore aimed at determining the frequencies of cysteine-cysteine receptor 5 (CCR5) genes V3 loop region of glycoprotein 120 in HIV subjects in Yenagoa.

Materials and methods

Study area

The study was conducted in Yenagoa and analyzed at the medical laboratory science department (Molecular laboratory) of the Niger Delta University, Amasoma both in Bayelsa.   Yenagoa is the headquarters of Yenagoa Local Government Area and also the capital of Bayelsa State. It is a Wetland located in Delta, South-south region of Nigeria. It is inhabited mostly by people of the Ijaw tribes. The official language is English but several other Ijaw dialects are also spoken in Yenagoa, some of the Ijaw dialects are: Epie-Atissa, Ekpetiama, Gbarian, Zarama and Biseni.  It has an area of 706 km² and a population of 352,285 according to the 2006 census with geographical location of 4°55′29″N 6°15′51″E.

They are mainly farmers and also engage in fishing. Some of their staple foods include cassava, plantain and rice.

Study population

HIV positive patients from the state owned hospitals with heart to heart centres were recruited for the study. Both male and female ages between 18 and 60 years

Determination of sample size

Test size was resolved utilizing G-power measurable programming, with a power of 80%. A specimen size of 100 has 80% power to identify an expansion of 9% with level (alpha) of 0.05 (two-followed). In this way a specimen size of 100 was utilized for the study.

 Experimental design

A cross-sectional observational study design was employed in this study. It was descriptive/diagnostic study. Subjects were randomly selected without bias.

Ethical consideration

Ethical approval for this study was received from the Bayelsa State Ethical Committee of the Ministry of Health. Subjects were educated on the objects, advantages and procedures of the study and were guaranteed the confidentiality of their data. All methods were performed in accordance with relevant guidelines and regulations.

Informed consent

Informed consent was obtained from all subjects and experiments were performed in accordance with relevant guidelines and regulations as enshrined in the Helsinki declaration, 2014. The study had no financial involvement on the part of the enlisted subjects.

 Inclusion criteria: All HIV positive patients between age 18 and 60 who consented to the study were included in the research.

Exclusion criteria: All HIV negative patient of any age where excluded from the study.

Collection of sample

Blood samples (4mls) were collected from each subject with normal stasis by venipuncture from either the antecubital vein or the dorsal vein under sterile condition and dispensed into tubes containing the anticoagulant ethylene diamine tetra acetic acid (EDTA). The specimens were labeled with the subject’s age, sex and identification number. The EDTA samples were kept in the refrigerator at a temperature of 2-8oC until processing.  

Molecular studies

DNA extraction

Genomic DNA was isolated from the EDTA anti-coagulated whole blood samples using Quick gDNA Mini Prep kits supplied by Zymos Research Inqaba, South Africa. Procedures for extraction was done according to the manufacturer’s instructions as shown below;

To a 1.5 ml micro-centrifuge tube, 50 µl of anti-coagulated whole blood was added, then 200 ul of Genomic Lysis Buffer was added. This was mixed completely by vortexing for 4-6 seconds and incubated at room temperature for 10 minutes. The mixture was transferred in a collection tube into a Symo-Spin and was centrifuged at 10,000 or 13,000 rpm for one minute and the collection tube was discarded with the flow-through. The micro-centrifuge tube was transferred into a new collection tube and 200µl of DNA Pre-Wash Buffer was added to the tube and was centrifuged for one minute at 10,000 or 13,000 rpm, 500 µl of Genomic DNA Wash Buffer was added to spin column and was centrifuged for 10,000rpm for one minute. The spin column was transferred into a clean micro-centrifuge to which was added 10 µl of DNA Elution Buffer. It was incubated at room temperature for 5 minutes and then centrifuged at high speed for 30 seconds to elute the DNA. The DNA was stored at – 200C for later use.

DNA quantification

This was done using the Thermo Scientific NanoDrop™ 1000 Spectrophotometer. One(1)µl of samples was placed on the pinhole of the nanodrop and the pedestral lowered to establish contact with the sample. The concentration of the DNA was read through the nanodrop software installed on a desktop computer.

Amplification of CCR5 gene

The amplification of the CCR5 gene was done on 2700 ABI thermal cycler in 20µl volume. The components were 2X master mix containing taq polymerase 1.5µl, dNTPs 200, 0.5µm of forward primers (P1(2975), 5´CAAAAAGAAGGTCTTCATTACACC-3  and reverse primers  P2(2976), 5´- CCTGTGCCTCTTCTTCTCATTTCG – 3´, and 3µl of DNA extract. The PCR condition were initially denatured at 95oC for 5min, 30 cycles of denaturation at 95oC for 30sec. Annealing at 55oC for 30sec and extension at 72oC for 50 sec and final extension at 72oC for 30min.

Amplification of the V3 region of Glycoprotein 120

The PCR reaction was done using a nested approach with the outer and inner primers 5’C2V3: TGTACACATGGAATTAGGCCA and 3’V3:

ATGAATTCATTACAGTAGAAAAATTCCC respectively. The second round of PCR was done using the primers J5’-2KSI: ATAAGCTTGCAGTGTAGCAGAAGAAGA and 3’C2V3: ATTTCTGGGTCCCCTCCTGAGG as outer and inner primer. All the reaction were on a final volume of 20ul with the following component , 2x master mix composing of taq polymerase dNTPs, mgcl, 0.2um of each primers. The conditions for the first rounds were as follows 95oC for 5min, 30 cycles of denaturation 95oC for 30sec. annealing 56oC for 30sec and extension 72oC for 1min with final extension at 72oC for 5min. The condition for the second round were similar for that of the first round except the annealing which was 52oC for 30sec .

The amplified products were resolved on a 1.5% agarose gel alongside a 100bp ladder and visualized on a UV trans-illuminator.

Sequencing of V3 region and CCR5 amplicons

Sequencing was done using the Big Dye Terminator kit on a 3510 ABI sequencer by Inqaba Biotechnological, Pretoria, South Africa. Sequences were subjected to bioinformatics analysis using MEGA 6.0 software.

Phylogenetic Analysis

Obtained sequences were edited using the bioinformatics algorithm Trace Edit, similar sequences were downloaded from the National Center for Biotechnology Information (NCBI) data base using BLASTN.  These sequences were aligned using Clustal X. The evolutionary history was inferred using the Neighbor-Joining method in MEGA 6.0 (11). The bootstrap consensus tree inferred from 500 replicates (12) is taken to represent the evolutionary history of the taxa analyzed. The evolutionary distances were computed using the Jukes-Cantor method (13).


Of the 100 subjects enrolled for the study, 27(27%) were males and 73(73%) were females. The age distribution of subjects showed that of the 27 males, 8(30%) and 19(70%) belonged to age bracket 40 -50 and 51-60 years respectively. Out of the 73 female subjects recruited, 4(5.5%), 16(21.9%), 41(56.2%) and 12(16.4%)  belonged to age bracket 18-28, 29-39, 40-50, 51-60 respectively. This is represented in Table 1 below.

Table 2 shows that all the subjects were homozygote for the CCR5 Gene. None were heterozygote or homozygote for the CCR5/Δ32 or Δ32/Δ32 mutant respectively.

The translation of protein sequence of various V3 region of the HIV isolates aligned using clustal X and translated to protein sequences on mega 6.0 showed few conserved amino acids among the HIV isolates  as shown in table 4.4 . This indicates polymorphism and strain variation among the HIV viruses in Yenagoa.

Agarose gel electrophoresis of the V3 region of GP 140 of the HIV Virus showed bands of 250bp when read against the 1kp molecular ladder.  S1-S10 represent the V3 bands, L1 represents a 100bp ladder while L2 represents a 1kb molecular marker. The V3 region of the HIV also showed bands of 300bp when run alongside a 100bp molecular ladder. This is shown in Figure 1

Figure 2 represents the agarose gel electrophoresis showing the CCR5 bands lane 1-10 represents the CCR5 gene bands. M and L represent the 100bp and 1kb ladder respectively.

The agarose gel of the CCR5 also showed single band of 189bp when run alongside a 100bp molecular ladder electrophoresis as shown in figure 2.

The phylogeny of the HIV isolates using  Neighbour Joining revealed that of the 17 HIV V3 regions successfully sequenced, 2(%) showed a highly relatedness to the indigenous HIV-1 isolate 01NGPL0567 from Nigeria, 1 (%) was related to the HIV isolate 05CVHAN14 from Cape Verde, 1(%), 1(%), 8(%), 3(%) and 1(%) related to strain J11243 from Saudi Arabia, strain NGIB.05_026 from Nigeria, strain TWG3.2 from Taiwan, strain IU6 clone G from Kenya and strain 280_10 from Cameroon respectively. (Fig 3)


Studies on the effects of CCR5 alleles in HIV disease progression have been made in different parts of the world (14, 17),. However, less data are available for this part of the world. There are reports of high frequencies of Δ32 alleles across different geographic regions in the world for which Caucasians ranked highest and Africans least (15, 16).  The highest recorded allele frequency is among the highly endogamous Ashkenazi Jews at above 22% (10). This study reported a 100% non-mutant CCR5 genotype for the study subjects. None was positive to the CCR5 heterozygous allele CCR5/Δ32 and none was also positive to the Δ32 homozygous deletion. Silva et al., (18) reported a 6.5% incidence of the heterozygous genotype among blood donors from São Paulo city, Brazil. Many studies have been performed to evaluate whether heterozygosity of the CCR5-del32 allele genotype affects the vertical transmission of HIV-1, or whether it affects disease progression (14, 19).

One meta-analysis has revealed that perinatal infection is not significantly altered by heterozygosity for CCR5Δ32 in children (20). Another meta-analysis has been performed among 10 studies including 1317 HIV-1 infected children, addressing the effects of CCR5Δ32. For progression to clinical AIDS, CCR5Δ32 showed an overall non-significant trend for protection (hazard ratio 0.84, 95% confidence interval 0.58-1.23). However, survival analyses showed a statistically significant time-dependence. The CCR5Δ32 genotype was associated with a decreased risk of death among perinatal infected children, although only during the first years of life (20).  Multiple factors may affect HIV-1 disease progression in perinatal-infected children. Such factors include in utero versus intrapartum infection, maternal disease status at the time of delivery (21), Therapeutic and prophylactic treatment of the mother and infant, and host human leucocyte antigen (HLA) genotype. In this study population, the CCR5 genotype was unable to account for the difference in pattern of disease progression among the three groups (rapid, moderate and slow progressors). However, we cannot exclude a potential role for these genetic characteristics, since sample size in our study was limited, and the allele frequency of CCR5- del32 was too low to allow statistical comparisons with adequate resolving power. Studies with larger populations may further elucidate the role of this allele and other host factor in the regulation of HIV-1 pathogenesis. This study also indicates polymorphism and strain variation among the HIV viruses in Yenagoa.


The CCR5 gene among HIV- positive individuals studied in Yenagoa is homozygous and there is no mutation. On the other hand, there is Single Nucleotide Polymorphism (SNP) of protein sequence in v3 region of p160 gene among HIV positive individuals in the study population. The study showed that the various strains of HIV virus among the studied population are CCR5 tropic.


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This work was carried out in the Molecular Biology Laboratory in the Department of Medical Laboratory Science, Niger Delta University, Wilberforce Island under the directorship of Prof Tatfeng Mirabeau. His pieces of advice and contributions in making this project a success is hereby acknowledged.

Additional Information

Competing Interests: The authors declare no competing financial or non-financial interests related to this work



Table  1: Demographic characteristics of the study participants

Age(years)             Male             Female Total

n=27              n =73 n=100


18-28   – 4(5.5%) 4(4.0)

29-39                           –            16(21.9%) 16(16.0)

40-50                        8(30%)            41(56.2%) 49(49.0)

51-60         19(70%) 12(16.4%) 31(31.0)


TOTAL          27(100%) 73(100%) 100(100)








Table 2: Performance charactristics of the Rapid Diagnostic Test used for HIV screening


Positive gold standard(100)

(V3 PCR)

Negative gold standard

(V3 PCR)




Positive by RDT

95 (True Positive)

6 (False Positive)

Negative by RDT

5 (False Negative)

94 (True Negative)





Sensitivity = 95%

Specificity = 94%

PPV = 94%

NPV = 94%

Prevalence = 0.9


Table 3: Distribution of CCR5 Genes among the Subjects




































































Table 4:  Protein Sequence of the V3 Region of the Various HIV Strains



        S1    S2   S3   S4   S5   L1   S6  S7    S8   S9  S10  L2



Fig 1   Agarose Gel Electrophoresis of the V3 Region  




Fig 2 The Agarose Gel Electrophoresis of the CCR5  


Fig. 3: Phylogenetic tree showing relationship between the HIV isolates

Percentage expression of Lewis B, Rh-e, N and ABO Antigens in descents of Bonny Kingdom, Rivers State, Nigeria.


Original Article

Percentage Expression of Lewis B, Rh-e, N and ABO Antigens in descents of Bonny Kingdom, River, State, Nigeria.

Ransom Baribefii Jacob*., Warmate, Tamuno-Eni, Beatrice Wobiarueri Moore-Igwe and Zaccheaus Awortu Jeremiah

 1Department of Medical Laboratory Science, Rivers State University, Port-Harcourt, Nigeria


*Corresponding Author:

Jacob, Ransom Baribefii

Department of Medical Laboratory Science,

Rivers State University, Port-Harcourt, Nigeria



Received: March 03, 2022, Accepted: April 21, 2022, Published: May 10, 2022



Introduction: The presence of rare blood group antibodies in the blood of donors/recipients stimulate red cell alloimmunization and constitute a major cause of transfusion related reaction after a compatible ABO cross match, yet remain one of the less routinely assessed prior to blood donation and transfusion in Nigeria. This cross-sectional study was carried out among indigenes of Bonny kingdom in Rivers State Nigeria to access the percentage expression of Lewis B, Rh-e, N and ABO antigens in descent of Bonny Kingdom Rivers State Nigeria.

Materials and Methods: One hundred and twenty (120) apparently healthy subjects consisting of sixty (60) females and sixty (60) males aged between 18-50 years all indigenes of Bonny Kingdom were recruited by structured questionnaire for the study. 4mls of venipuncture blood was aseptically collected from each participant into vacutainer bottle containing 0.5 mL of 1.2 mg/mL dipottasium ethylenediaminetetraacetic acid anticoagulant and a 5% cell suspension was prepared and used to determine the various blood groups antigens using the standard tube Agglutination technique.

Results: Results obtained shows a total of 94.1% (113) of the participants expressed Rh-e antigen, 45.8% (55) male and 48.3% (58) females. 7.5% (9) of the participants expressed the N antigen, 3.3% (4) male and 4.2% (5) females. 4.2% (5) of the study population expressed Lewis B antigen, 2.5% (3) males and 1.7% (2) female. The ABO system showed 68.5% (79) O blood group, 29.1% (35) male and 36.7% (44) female. 22(18.3%) typed for A blood group, 11.6% (14) male and 6.7% (8) female.  For B blood group 14.2% (17) persons were typed, 7.5% (9) male and 6.7% (8) female while 1.6% (2) persons were typed as AB and they are all females.

Conclusion: Rh-e phenotype occurred highest among the study participants. The proportion of the population without the expression of these antigens of the blood group tested has the potential to be alloimmunized during blood transfusion and develop antibodies, some of which can be responsible for transfusion reactions and haemolytic disease of the new born. This study has provided baseline data on the distribution of some blood group antigens in the Bonny Kingdom of Rivers State in Nigeria. Based on the finding in this study, it is recommended that Rh-e grouping be carried out on pregnant mothers, blood donors and recipients before transfusion; while Lewis B and N blood groups may be subjected to expansive population testing.

KEYWORDS: Alloimmunization, Lewis, Rh-e, N antigens, Bonny Kingdom.



The presence of rare blood group antibodies in the blood of donors/recipients stimulate red cell alloimmunization and constitute a major cause of transfusion related reaction after a compatible ABO cross match, yet remain one of the less routinely assessed prior to blood donation and transfusion in Nigeria and particularly in Bonny Kingdom (1).

Detection of blood group antibodies in a population and making available corresponding compatible negative blood group is a sure way of preventing red cell alloimmunization and transfusion related reactions both in pregnant mothers, blood donors and recipients. The Bonny Kingdom, otherwise known as Grand Bonny is a traditional state in the town of Bonny, located in Bonny Local Government Area of Rivers State, Nigeria. In the pre-colonial period, it was an important slave trading port and later was a key location for trading palm oil products. During the 19th century the British became increasingly involved in the internal affairs of the kingdom, and in 1886 assumed control under a protectorate treaty (2). 

In human plasma, Lewis antigens are found to be attached to erythrocytes, platelets and lymphocytes that are circulating by direct insertion of their lipid anchor into the plasma membrane of the above mentioned cells; while in body secretions, Lewis blood group antigens are also similarly attached to an amino acid component of the glycoprotein (3, 2). The Lewis antigens do not have their synthesis on the red blood cells, but are absorbed from plasma (3). 

Lewis antibody Leb occur as IgM in nature, it react at room temperature and can activate complement and does not cause haemolytic transfusion reaction or haemolytic disease of the newborn thus not clinically significant (3,9,2).

 The Lewis agglutinogens, are biochemical structures synthesized by exocrine epithelial cells that are absorbed passively into red blood corpuscular bi-layers, and some group of Lewis agglutinogens function as counter ligands for selectins, this has been observed to be consistent with the relationship of Lewis antigens in the occurrence or development of thrombosis. Lewis-b (Leb) is a receptor for Helicobacter pylori (5).

Research has shown the frequency distribution of Lein Whites and in Blacks as 72% and 55 % respectively (6, 7, 8). Although the Lewis antigens are weakly expressed in cord blood, their expression in human begins at 2 years old (Okoroiwu et al., 2018). An earlier study on the prevalence of Lewis A amongst indigenes of Bonny Kingdom showed that a total of 12 (10%) of the study population were positive for Lewis A blood group antigen, 3(2.5%) females and 9(7.5%) males (2).

The Rh blood group system consists of 49 defined blood group antigens, among which the five antigens D, C, c, E, and e are the most important (9,10). Anti-D, anti-C, anti-E, and anti-e have all been involved in haemolytic transfusion reactions, particularly delayed reactions (11, 1, 2). The clinically important Rhesus antigens C, c, E and e are the result of RhCE protein changes at only five amino acid locations (12, 13, 14). 

Report in literatures shows that the Rh antigen C, c, E, and e are not so immunogenic but are highly important in patient care upon the development of the corresponding antibody (2). Beside their importance in blood transfusion and haemolytic disease of the newborn, the physiological function played by this protein can only be speculated as been involved in the transportation of ammonium across the RBC membrane and the maintenance of the integrity of the red cell membrane (15).

The MNS nitrogen system is a human blood group system based upon two genes (glycophorin A and glycophorin B) on chromosome 4 (10). There are currently 46 antigens in the system but the five most important are called M, N, S, s, and U (16, 10). The MN blood group system is under the control of an auto social locus found on chromosome 4, with two alleles designated Lm and Ln. The blood type is due to glycoprotein present on the surface of red blood cells, which behaves as native nitrogen. Phenotypic expression at this locus is codominant because an individual may exhibit either one or both antigenic substances. Frequencies of the two alleles vary widely among human populations (17).

Research shows that the anti-N is sometimes seen in dialysis patients, due to cross-reactions, with the residual formaldehyde from sterilizing the equipment. This is usually irrelevant in transfusion since this variant of the antibody does not react at body temperature (18).

Following paucity of information on the occurrence and percentage distribution of Lewis B, Rh-e, N blood group antigen amongst descents of Bonny, it is therefore, necessary to carry out serological identification of Lewis B, Rh-e and Nantigens in other to access the percentage expression of these antigens are in the population. This will enable medical Scientist, Blood bank facilities within the kingdom to possibly associate their occurrence with disorders (especially haemolytic disease of the newborn, transfusion related reactions), project for the stocking of negative compatible blood for transfusion. This work is thus aimed at determining the percentage expression of Lewis B, Rh-e, N and ABO blood group antigens in descents of Bonny Kingdom Rivers State Nigeria.

Materials and Methods

Study Design and Population

This cross-sectional study involved one hundred and twenty (120) apparently healthy subjects consisting of sixty (60) females and sixty (60) males aged between 18-50 years all indigenes of Bonny Kingdom recruited by structured questionnaire for the study.

Four millilitres (4mls) of Venepuncture blood sample was obtained aseptically from the antecubital fossa of each participant with the use of vacutainer containing 0.5 mL of 1.2 mg/mL of dipotassium ethylene diamine tetra-acetic acid (EDTA) and mixed by gentle tilting for the serological determination of Lewis B, Rh-e, N and ABO blood group antigens respectively.

Materials and Methods

Determination of ABO Blood Group Using Tube method


Red blood cells were phenotyped for A, B, AB and O antigens according to standard serologic protocol (tube method).

Determination of Lewis-B, Rh-e and N Blood Group Antigen Using Anti-leb, Anti Rh-e and Anti- N Monoclonal, Lorne Laboratories Microtitre Agglutination Techniques

Phenotyping of red cells was done using Micro-titre Agglutination technique as describe by Lorne laboratory Ltd. A 5% suspension of red blood cell was prepared using normal saline. 20ul of anti-Leb, anti-Rh-e and anti-N antibodies were added unto separate micro-titre plate, and 20ul washed red cell was added into the micro-titre plate containing the anti-Leb, anti Rh-e and anti-N antibodies. The sample was incubated for 15minutes with intermittent rocking and observation for agglutination every 30 seconds. If no agglutination found after 30miutes 20ul of LISS antibody was added and observed for 15-30 minutes, if no agglutination, the sample was placed in a slide under the microscope and examine microscopically for agglutination. Presence of agglutination indicates a positive result and absence of agglutination indicates negative result.

Data collected was statistically analysed by simple percentage calculation and data presented in Tables.


The study population consisted of a total of 120 apparently healthy Bonny indigenes, 60 males and 60 females aged between 18-50 years. 60 (50%) males and 60 (50%) females as shown in Table 1. Frequency distribution of ABO blood group amongst the Study population is shown in Table 2. 22(18.3%) [14(11.6%) males and 8(6.7%) females] typed positive for A antigens, 17 (14.2% [9(7.5%) males and 8(6.7%) females typed for B antigens, 2(1.6%) all females typed for AB antigens and 79(65.8%) 35(29.1%) male and 44(36.7%) typed for O antigens in the population. The frequency distribution of Rh-e Blood Group amongst the study population is shown in table 3 One hundred and thirteen (113) subjects from the total population (120) had Rhesus-e blood group which represent 94.17%. Out of the total population, fifty five (55) subjects who had Rh- e blood group were male while fifty eight (58) subjects were female. This represents 45.83% and 48.33% respectively. For the N Blood Group 7.5% frequency was obtained (9) subjects from the total population studied. Of the 9 persons four (4)  were males while five (5) were females representing  3.3% and 4.2% respectively. Five (5) subjects from the total population had blood group Lewis B. This represents 4.16% and of this, three (3) were males while two (2) were females thus representing 2.5% and 1.7% respectively (Table 3)


Table 1 Demographic Data of study population

Gender of Subjects

Number of participants

Frequency (%)











Table 2 Frequency Distribution of ABO Blood Group Amongst the Study Population

ABO antigens/Gender

Number of participants

A-antigen (%)

B-antigen (%)

AB- antigen (%)

O- antigen (%)



14 (11.6%)

9 (7.5%)

0 (0%)

35 (29.1%)



8 (6.7%)

8 (6.7%)

2 (1.6%)

44 (36.7%)



22 (18.3%)

17 (14.2%)

2 (1.6%)

79 (65.8%)


Table 3 Frequency Distribution of Rhesus e, N, & Lewis b, Blood Groups amongst the Study       Population

Subjects/ Blood groups


Number of participants

Frequency (%)



55 (45.8)



58 (48.3)



113 (94.2)

N blood Group





4 (3.3)



5 (4.2)



9 (7.5)

Lewis b





3 (2.5)



2 (1.7)



5 (4.2)



This is a cross-sectional study carried out among indigenes of Bonny kingdom in Rivers State, Nigeria to assess the frequency distribution of Lewis B, Rhesus e, N and ABO blood groups amongst Bony indigenes.  One hundred and twenty apparently healthy subjects consisting of sixty (60) females and sixty (60) males, age between 18 to 50 years participated in this study.

From the result obtained, it was shown that five (5) subjects, three (3) males and two (2) female (i.e 2.5% and 1.67%) respectively from the total population were positive for Lewis B blood group. This represents 4.16% of the total population. This finding is lower than the report of Christian et al., (19) who in their study carried out amongst one hundred (100) Ogoni indigenes, reported a percentage distribution of 11.88% in the total population with a frequency occurrence of 12 for Lewis B (Leb) blood group. This is also in deviant from the percentage distribution of 23 % as reported by Lorne Laboratories, (20), amongst African-Americans and also not in tandem with Reid et al. (21), who reported a high percentage distribution of 55% amongst Blacks. The result obtained can be due to the population size used in the study and the peculiarity of the population under study.

The result for Rh-e antigens shows that one hundred and thirteen (113) subjects from the total population had Rh-e blood group. This represents 94.2%. Out of the total population; fifty five (55) subjects were male while fifty eight (58) subjects were female representing 45.83% and 48.33% respectively. This pattern of result is in discord with a study by Christian et al. (8). In their study carried out amongst one hundred and one (101) Ogoni indigenes, the Rh-e blood group distribution amongst the study, revealed a percentage distribution of 25.74%. The finding in this study is also in contrast with Reid et al. (21) who reported a 21% Rh-e percentage distribution amongst Indians. The high prevalence reported seen in this study can be related the peculiarity of the race/population under study and also due to the high rate of blood transfusion practices, blood donation and possibly exposure from pregnancies amongst the population.

A total of nine (9) subjects from the total population had N blood group, representing 7.5% of the total population, four (4) males and five (5) females representing 3.3% and 4.17% respectively. These observations were at deviant from the frequencies in the Eastern Province of Saudi Arabia as reported by Owaidah et al (10) who reported the frequencies of the MNS antigens as follows: M antigen (87%), N (52%), S (59%), and s (83%) in one hundred Saudi arabia blood donors. Also finding in this research was not in tandem with the research of Hallawani et al (22) who found a frequency expression of 51.67% in 149 randomly samples anonymous Saudi blood donors living in Jazan Province southwestern Saudi Arabia. Research shows that the anti-N is sometimes seen in dialysis patients, due to cross-reactions with the residual formaldehyde from sterilizing the equipment although this is usually irrelevant in transfusion since this variant of the antibody does not react at body temperature (18). However, the low prevalence seen in this research could be attributed to the peculiarity of the race/population under study, the fact that subjects in this population may not have been exposed to dialysis as compared to other study population in literatures and also due to the small number of samples used in the study.

The ABO blood group system percentage expression amongst studied population revealed  a total of 22 (18.3%) individuals expressed the A-antigen, 17(14.2%) individual expressed the B-antigens, 2 (1.6%) all females expressed the AB-antigens while 79 (65.8%) of the total population expressed no A or B antigens thus representing the O-blood group. This is in agreement with a study by Erhabor et al., (23). In their study, they recorded similar pattern of distribution in ABO/Rh blood group in students of Niger Delta University. Bakare et al. (24) recorded the similar pattern of ABO blood groups distribution in Nigerian Ethnic groups. The blood group O of the ABO system is thus the most highly expressed amongst the study population and in almost all the population and ethnic groups as revealed in literatures.


Rh-e phenotype occurred highest among the study participants. The proportion of the population without the expression of these antigens of the blood group tested has the potential to be alloimmunized during blood transfusion and develop antibodies, some of which can be responsible for transfusion reactions and haemolytic disease of the new born. This study has provided baseline data on the distribution of some blood group antigens in the Boony Kingdom of Rivers State in Nigeria. Based on the finding in this study, it is recommended that Rh-e grouping be carried out on pregnant mothers, blood donors and recipients before transfusion; while Lewis B and N blood groups may be subjected to expansive population testing. 



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  2. Jacob, R.B., Akah, M.N., Robinson-Mbato, L., Christian, S.G., Olusola, O.A., and Eze, E.M. Expression of Kidd, Rh-C, E, and D Antigen among Indigenes of Bonny Kingdom, Nigeria. Journal of Blood Disorders and Transfusion. 2022; 13:492.
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  8. Christian, S. G., Eze, E. M. & Essor, J. E. ABO, Rhesus blood groups and haemoglobin variants distribution among individuals with Helicobacter pylori in Igwuruta-Ali, Rivers State. Journal of Advances in Medicine and Medical Research,2019; 28(10): 1-8.
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Role of microRNA in acute Myeloid Leukaemia


Review Article

Role of MicroRNA in Acute Myeloid Leukaemia: An Overview

Aruomaren Austin Iroghama1, 2 and Ude Arinze2

1Department of Medical Laboratory Science, School of Basic Medical Sciences, University of Benin

2Biomedical Science Department, University of the West of England, Bristol, UK

*Corresponding Author

Aruomaren Austin Iroghama

Department of Medical Laboratory Science, School of Basic Medical Sciences, University of Benin

Email: iroghama.aruomaren@uniben.edu 


Received: February 28, 2022, Accepted: April 08, 2022, Published: May 10, 2022



Expression of MicroRNAs (miRNAs), key regulators of normal haematopoiesis, is reportedly dysregulated in acute myeloid leukaemia (AML).Aberrant microRNA expression has been associated with different subtypes of AML, where they exert tumor suppressive or oncogenic functions. Deregulated microRNA expression has been shown to be of prognostic significance. Furthermore, it has been demonstrated that repression and ectopic expression of microRNAs affect response to treatment in AML. Consequently, microRNAs may act as potential biomarkers in AML hence production of new anti-AML drugs directed towards increasing and/or silencing miRNA expression may suffice.

Keywords: microRNA, AML, Tumour suppressor gene



MicroRNAs (miRNAs) are short non-coding 19-25 nucleotide RNAs that control gene expression by cleaving their target messenger RNA (mRNA) thus inhibiting protein translation. These miRNAs are produced in the nucleus and cytoplasm (Figure 1.1), where they undergo successive enzymatic cleavage by RNA polymerase II, DROSHA and DICER1from primary transcripts (pri-miRNA) through hairpin precursors miRNA  (pre-miRNA) to mature miRNA before integration into the RNA-inducing silencing complex (RISC) [1,2].

MicroRNAs play significant roles in haematopoiesis; regulating it by targeting various factors involved in cell proliferation, differentiation and apoptosis. However, aberrant microRNA expression has been linked to pathogenesis of varied diseases, including cancer. Recently, aberrant microRNA signature was recognized as one of the hallmarks of cancer [2]. Linkage between microRNA and cancer was first reported in chronic lymphocytic leukaemia (CLL) [3]. Several methods, including quantitative reverse transcription polymerase chain reaction (qRT-PCR), have enabled validation of individual microRNA expression to clarify the linkage of aberrant microRNA expression to cancer, whereby microRNAs may act as oncogenes or tumor suppressors [2]. Upregulation of oncogene microRNA (oncomiRs) and downregulation of tumor suppressor microRNA support leukemogenesis.

Acute myeloid leukaemia (AML), a heterogeneous haematological malignancy, travels fast predominantly resulting in poor outcome. Cytogenetic abnormalities, age, chemotherapy and secondary haematological disease affect prognosis of AML. Distinct microRNA expression signatures in subtypes of AML suggest great potential to use these miRNAs as biomarkers.


Figure 1.1 MicroRNA biosynthesis pathway. Primary miRNA (pri-mRNA) is transcribed by RNA polymerase II (RNA pol II) in the nucleus. Pri-miRNA are cleaved by DROSHA and its cofactor DGCR8 (DROSHA-DGCR8complex) producing precursor miRNA (pre-miRNA). Pre-miRNA is transported by Exportin-5 protein into the cytoplasm, where they are cleaved by DICER1 before incorporation into the RNA-induced silencing complex (RISC). Mature miRNA binds to 3’-untranslated region (3’-UTR) of a target mRNA to induce mRNA cleavage, translational repression and/or mRNA deadenylation [2].


Biomarkers are biochemical/genetic/molecular features that indicate physiological state of an individual and also offer prognostic, diagnostic and/or therapeutic information in diseases. Distinct microRNA expression may offer information on diagnosis and prognosis of AML to better the clinical outcome of patients [1,4]. The prospective use of miRNAs as biomarkers may further provide novel therapeutic targets in AML. This review focuses on the potential role of specific miRNAs as biomarkers in AML with reference to recent findings.


These are single-stranded RNA molecules that regulate various physiologic processes in the body. There are about 2042 microRNAs in microRNA registry [5]. Biosynthesis of miRNAs is illustrated in Figure 1.1.


AML is a malignant disease caused by increased proliferation and differentiation block in myeloid blasts. Its incidence increases with age; the median age of AML is 68 years. Cytogenetic abnormalities and dysregulated gene expression contribute to the heterogeneity of AML pathogenesis. The major prognostic factor in AML is leukaemic karyotype. Based on French-American-British and World Health Organization classifications, AML is grouped into various subtypes and associated risk depending on cytogenetics (Table 3.1).

Table 3.1: Cytogenetic classification of various subtypes of AML based on prognosis

Cytogenetic abnormality

Subtype of AML

Associated risk/prognosis

Reference (s)


Core binding factor leukaemia (CBFL)


6, 7

RUNXI/MECOM (EVI1) t(3;21)

Core binding factor leukaemia (CBFL)



CBFB-MYH11 inv(16) or t(16;16)

Core binding factor leukaemia (CBFL)


6, 7

PML-RARA t(15;17)

Acute promyelocytic leukaemia


2, 6, 7

Trisomy 8

AML not otherwise specified


7, 8

Translocation t(9; 11)

Acute monocytic leukaemia



Brain and acute leukaemia cytoplasmic (BAALC) expression

Cytogenetic-normal AML (CN-AML)



Nucleophosmin (NPM1)

Cytogenetic-normal AML (CN-AML)


9, 10

Internal tandem duplication of fms-related tyrosine kinase gene (FLT3/ITD)

Cytogenetic-normal AML (CN-AML)



CCAT/enhancer binding protein alpha (C/EBPA)

Cytogenetic-normal AML (CN-AML)


1, 2


Cytogenetic-normal AML (CN-AML)


4, 9


Core binding factor leukaemia (CBFL)



Deletion -7q

AML not otherwise specified



Deletion -7

AML not otherwise specified


6, 7

Translocation t(6;9)

Acute myeloblastic leukaemia



ERG expression

Cytogenetic-normal AML (CN-AML)



Translocation t(9;22)

Philadelphia Chromosome positive-AML



MN1 expression

Cytogenetic-normal AML (CN-AML)



Deletion -5

Therapy-related AML



Trisomy 21

AML related to down’s syndrome



Cyclic-AMP responsive element binding protein (CREBBP)

Therapy-related AML



Trisomy 13 (Patau syndrome)

AML, minimally differentiated (M0)



Translocation t(1; 22)

Acute megakaryocytic leukaemia (M7)



Deletion -5q

Therapy-related AML



11q23 abnormalities

Acute monocytic leukaemia (M5)


7, 8, 11

Wilms tumor 1 (WT1)

Cytogenetic-normal AML (CN-AML)



This is a combination of FAB and WHO classifications of AML.



Due to the heterogeneity of AML, diagnosis and prognosis are a conundrum, however, the choice of treatment depends on cytogenetic information. MiRNAs have been shown to be differentially expressed in normal and AML cells, thus highlighting a possible role as biomarkers in AML [5, 9]. However, comparative studies of microRNA signatures expressed on normal and AML cells indicated an overlap among different studies. This disparity may be ascribed to differences in samples collected, sample collection methods, profiling methods, sample/control size and heterogeneity of patients [1, 2]. Table 3.1 shows a myriad of miRNAs that have been linked with pathogenesis of AML.

Table 4.1 MicroRNAs in various subtypes of AML

AML Subtype

MicroRNA(s) involved


Reference (s)









Promotes apoptosis by targeting anti-apoptotic protein Pim-1

Promotes differentiation by targeting HOXA10/MEIS

Blocks nuclear transcription factor NF-1A to promote apoptosis and inhibit proliferation

Inhibits differentiation of myeloid cells by targeting CEPBB, NFkB, JUN, PU.1, SHIP1

2, 3, 4, 6, 10, 12, 13, 14.

Inv (3) RPN-MECOM (EVI1)


Promotes apoptosis through negative regulation of NOTCH1 and BCL2



KIT overexpression


Forms a network with SP1/NFkB1/HDAC resulting in KIT expression




ERG expression



Both miRNAs regulate ERG




BAALC overexpression



Negatively regulates BAALC expression

Targets USP40 and FBXL20 genes and is associated with poor overall survival (OS)

1, 2



CREBBP overexpression


Targets CREBBP regulating its expression







Targets pro-survival proteins BCL2STAT5, JAK2

Regulates p53 through involvement of erythroid transcription factor GATA-1

2, 8


Acute megakaryobkastic leukemia



Reduces expression of ST18 and DICER1

Associated with poor prognosis


2, 15


NPM1 mutation







Targets IRF2 and KIT


Targets MN1,CLCN3, CRKL

Anti-apoptotic; targets KLF4 and RB1CC1

1, 2, 3, 14

CEBPA mutation





Erythroid differentiation of leukemic blasts

Causes E2F3 overexpression and  cell proliferation

Reduces expression of CEBPA suppressor NF1-A and transcription factorE2F1

1, 2






Promotes apoptosis by targeting BCL2

Involved in malignant transformation

Promotes angiogenesis

Promotes macrophage/monocyte differentiation


t(8;21) AML1/ETO







Regulates myeloid differentiation; epigenetically regulated by RUNX1/RUNX1T1

Regulates tumour suppressor PLK2 and inhibits apoptosis

Promotes myelopoiesis and apoptosis

Inhibits erythropoiesis through KIT downregulation


2, 3, 16, 17






Enhances proliferation and subdues apoptosis through regulation of pro-apoptotic protein, BAK1

Targeted by PML-RARA

Targeted by PML-RARA


t(8;16) (p11;p13) and KAT6A-reaarangement





Targets proto-oncogene RET that encodes tyrosine-kinase receptor

Targets anti-apoptotic protein, BCL2

2, 6

Trisomy 8




Targets myeloid transcription factor CEBPA









Same as in t(8;21) AML1/ETO



Targets DNMT3A, DNMT3B and TCL1

2, 3

AML, not otherwise specified





Targets SP1, DNMT3A, DNMT3B, TCL1, TET1, CDK

Targets CCNT2, CDK6

Targets CCNT2, CDK6

2, 21.


t(11q23) MLL-rearranged AML

















Associated with poor prognosis in both ALL/AML

Enhances myeloid differentiation through MYB gene

Regulates E2F2, NF1A, PU.1 Involved in myeloid differentiation


Regulated by MLL fusion protein


Described as a lymphoid-specific microRNA

Targets TCL-1, MCL-1

Disrupts cell cycle by targetingCDK inhibitor

Targets tumor suppressor PTEN

Targets TGFβ1-regulator SMAD1

Targets homebox genes, HOXA7, HOXA9, HOXA11, PBX3

Represses HOXA9 cofactors, MES1 and PBX3

2, 4, 8, 10, 13, 14, 15, 17, 18

↑, upregulated – oncomiR; ↓, downregulated – tumor suppressor.


Tumor suppressor microRNAs are microRNAs that repress the action of oncogenes that enhance leukemogenesis. These microRNAs promote differentiation and apoptosis of myeloid cells hence they are epigenetically silenced by oncogenes in AML. Several studies have illustrated a negative correlation between these miRNAs and aggressiveness of AML [8, 15].


This family consists of miR-181a and miR-181b. MiR-181a was the first microRNA to be independently linked to prognosis in AML. In 2010, Schwind et al. [9] revealed an association between miR-181a overexpression and favourable outcome in 187 de novo CN-AML patients, especially in patients with FLT3-ITD and/or NPM1wild-type mutations. The prognostic significance of miR-181a overexpression in AML has been validated by two other studies. Recently, miR-181a overexpression was reportedly linked to the favourable prognosis group [15] whilst Li et al., [20] reported that upregulation of miR-181a and miR-181b in two sets of cytogenetically abnormal (CA)-AML patients was associated with better prognosis and longer overall survival (OS). Furthermore, miR-181a upregulation in AML cell line enhanced sensitivity to AML drug, Ara-C by inducing apoptosis of drug-resistant leukemic cells [19]. This correlates with Li et al. [20] findings of increased apoptosis and decreased proliferation in AML cells and mouse models following miR-181a and miR-181b overexpression.

Genes involved in development processes and innate immunity have been inversely linked to miR-181 expression. These genes include, toll like receptors (TLR2/TLR4), interleukin pathway (CASP1, IL1β, IL1RN), transcription co-regulator ID1, FL1 gene, transcription factor TCF4, anti-apoptotic protein BCL2, homebox genes (HOXA/HOXB) andHOX cofactors, MEIS1 and PBX3 [8, 9, 19]. These genes promote leukemogenesis and are adverse prognosticators in AML.

However, miR-181 overexpression is directly associated with haematopoietic differentiation promoter TCF3 gene expression, decreased NF-kB expression, decreased miR-155 (oncomiR) expression, high haemoglobin level, white blood cell (WBC) count, percentage of circulating blasts and absence of extramedullary infiltration [9, 13, 15].These findings suggest miR-181 regulate immune response, differentiation and apoptosis hence highlighting a possible role of miR-181 as biomarkers in AML.


The lethal-7 (let-7) family consists of ten isoforms. Let-7a is the most studied member of this family. Conflicting reports suggest let-7a may act as a tumor suppressor or oncomiR in leukemogenesis.

Recently, Li et al. [20], reported that let-7a-3 overexpression in 102 newly diagnosed AML patients compared to normal patients is associated with a poor outcome, in contrast to what their results reveal (Figure 4.1). However, let-7a expression was repressed by stromal derived factor (SDF)-1α–mediated CXCR4 activation in primary AML cells whereas transfection of let-7a induced inhibition of CXCR4and increased sensitivity of AML cells to Ara-C both in vitro and in vivo [24]. Further ChIP assay showed that transcription factor, Yin Yang 1 (YY1) binds to let-7a following SDF-1α/CXCR4 signalling to thwart its suppressive actions. Genes involved in the regulation of apoptosis and immune responses, such as BCL-XL, CDK5, MYC, CASP3, RAS and interleukin-16 are possible targets of let-7a [24].

Other let-7 family members have also been associated with pathogenesis of AML. Several let-7 family members were upregulated in AML patients with NPM1 mutation and 3q26 abnormalities respectively [3,14]. Also, circulating levels of let-7b and let-7d were upregulated and downregulated respectively in plasma of 20 AML patients when compared to plasma of normal controls [5].


Figure 4.1: Overall (A) and relapse-free survival (B) of AML patients with and without miR-let-7a-3 expression [20].


Upregulated let-7b expression can also distinguish acute leukemic types in mixed-lineage leukemia (MLL) which has poor prognosis [15]. These data suggest let-7 may be of prognostic and diagnostic significance in AML, however, the role of let-7a has to be elucidated with a large-cohort study.


The miR-29 family includes three members often seen in 2 clusters: miR-29b-1/miR-29a and miR-29b-2/miR-29c [1]. MiR-29a may be overexpressed or under expressed in AML, depending on the molecular abnormalities. MiR-29a is overexpressed in patients with NPM1 and/or without FLT3/ITD mutations [6, 12] whereas Wang et al. [17] reported miR-29a under expression in 10 newly diagnosed AML patients.

Recently, diagnostic significance of miR-29a downregulation was revealed. Wang et al. [21] reported combined downregulation of miR-29a and miR-142-3p in peripheral blood mononuclear cells (PBMC) in 52 AML patients offered a better diagnostic outcome.

MiR-29b, on the other hand, is downregulated in AML, especially MLL due to its regulatory function in MLLT11 expression [17, 21]. Inverse correlation between miR-29b and MLLT11 expression was reported in primary AML cells in vivo and in vitro [25]. The authors further associated miR-29b downregulation with poor OS in MLL, in connection to Wang et al. [17] report that overexpression of 3-miRNA-outcome (miR-29b, miR-26a, miR-146a) signature in 40 AML patients conferred good prognosis.

Proto-oncogenes involved in haematopoietic development have been expressed as direct targets of miR-29. These genes include, MCL1, TCL1, DNTM3A/B, CDK6, JAK2, IGFR, SALL4 and HOXA9 [6, 21, 25]. These data reveal miR-29 offer some prognostic information due to their role in differentiation and apoptosis.


MiR-150 is mainly expressed in secondary lymphoid organs and is over expressed during lymphoid development. It also promotes myeloid differentiation and is down regulated in AML [18]. Discordant miR-150 expression in different cells enables it distinguishes between acute leukemic types in MLL [11, 17]. Furthermore, combined plasma levels of miR-150 and miR-342 were found to be similar in AML patients that achieved complete remission (CR) and healthy controls [5].

The differentiate effect of miR-150 means it impairs proliferation of myeloid cells in AML. Therefore, miR-150 directly targets oncogene MYB that promotes self-renewal of cells [18]. These findings suggest miR-150 may have diagnostic and prognostic effect in AML.


Oncogene miRNAs (oncomiRs) are miRNAs that promote leukemogenesis by suppressing tumor suppressor genes. In AML, oncomiRs expression is upregulated by leukemic stem cells thus affecting clinical outcome of patients. Several oncomiRs associated with AML have been identified (Table 3.1).


MiR-155 is often overexpressed in acute leukemia. MiR-155 is regarded as a lymphoid-specific microRNA that targets PU.1, JUN and C/EBPβ genes thus inhibiting differentiation of myeloid cells [3, 17]. However, miR-155 overexpression is often associated with FLT3/ITD mutation in AML [3, 6, 7, 10, 13, 14, 15]. This association may be clinically relevant due to the prognostic value of FLT3/ITD and classification of miR-155 as an oncomiR. These findings were supported by miR-155 repression by anti-leukemic activity of silvesterol in AML cell lines carrying FLT3/ITD mutation [22].

Interestingly, miR-155 is also downregulated in FLT3/ITD-expressing AML cells. MiR-155 expression was low in FLT3/ITD-expressing murine myeloid FDC-P1 cells [12]. This may due to independence of miR-155 expression on FLT3/ITD signaling [14]. These data suggest miR-155 overexpression is not specifically linked to FLT3/ITD mutation, therefore therapeutic targeting of miR-155 in FLT3/ITD-AML may offer no solution. New studies should be done to determine the prognostic and/or diagnostic significance of aberrant miR-155 expression in AML.


MiR-9 has three isoforms, namely miR-9-1, miR-9-2 and miR-9-3. Its role in haematopoiesis is unknown due to contrasting reports. MiR-9 overexpression is synonymous with AML and may affect prognosis in AML. A recent study revealed miR-9 overexpression in a heterogeneous cohort of 101 newly formed AML patients had a negative impact on OS and RFS [23]. Similarly, ectopic expression of miR-9 promoted MLL-AF9/HOXA9-mediated transformation in normal mouse BM progenitor cells and in vitro [24]. Further ChIP assay in AML cell line showed that MLL-AF9 fusion protein binds to promoter regions of miR-9 to enhance its overexpression.

However, ectopic expression of miR-9 in ectopic viral integration site I (EVI1)-induced AML promoted myeloid differentiation and apoptosis in a murine model whilst EVI1 repressed miR-9 expression by binding to miR-9-3 promoter [16]. Furthermore, miR-9 is also downregulated in AML1/ETO rearrangement [3].

These findings suggest role of miR-9 in AML depends on the subtype involved. This disparity could be attributed to its isoforms. Gene-expression profiling revealed miR-9 targets separate genes in both context. When downregulated, miR-9 targets inhibitors of myeloid differentiation, Fox01 and Fox03, but targets RHOH and RYBP when upregulated [13, 24].


Figure 5.1 MicroRNA-based therapeutic strategies.AMiRNA mimics can reverse the expression and action of endogenous tumor suppressor miRNA. BGene therapy can either silence or increase expression of tumor suppressor miRNA through vector-driven expression of pre-miRNA/pri-miRNA and shRNA/siRNA respectively.MicroRNA-masks compete with oncomiRs for antisense binding to mRNA target without inducing mRNA degradation. DAntimiR silence oncomiRs and inhibit repression of tumor suppressor gene expression by binding to oncomiRs. ESponges or decoys contain miRNA binding sites that block binding of overexpressed oncomiRs to their target sites. ORF, open reading frame. TSG, tumor suppressor gene [2].


Noting the abundance of the aforementioned information, it is evident microRNA may act as biomarkers and provide a novel insight into AML therapy. The fact that miRNAs can differentiate acute leukaemic types and enhance drug sensitivity suggest their potentials as therapeutic targets in AML. The development of microRNA-based therapy (Figure 5.1) is aimed at increasing the level of tumour suppressor microRNAs and/or silencing oncomiRs expression.


AML travels a very fast course hence accurate diagnosis and prognosis is fundamental to give the clinician a timeframe in the treatment of AML patients. Despite the success of stem cell transplantation, high relapse rates and early death are associated with the AML phenotype due to its heterogeneity. The linkage of aberrant microRNA expression to pathogenesis, diagnosis and prognosis of AML suggests a possible role for microRNAs as biomarkers as clinicians seek a solution to this problem. MicroRNAs are involved in regulation of distinct physiological processes, such as gene expression and haematopoiesis. These microRNAs offer novel therapeutic targets and suggest the development of new microRNA-based leukaemic therapies with minimal side effects. However, large-scale randomized controlled trials should be conducted to validate the efficacy and bioavailability of potential microRNA-based drugs in AML


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