Vol 1, No 2 & 3, september 2022

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

Posted onSeptember 30, 2022May 30, 2023Leave a comment

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African Journal of Laboratory Haematology and Transfusion Science

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

Volume 1, Number 2 & 3, September, 2022

ISSN: 2814-0591 (Print)

ISSN: 2814-0605 (Online)

EDITORIAL BOARD MEMBERS

EDITOR-IN-CHIEF

Prof Teddy Charles Adias

Federal University Otueke, Bayelsa State, Nigeria

teddyadias@yahoo.com

DEPUTY EDITORS
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
EDITORIAL ADVISERS
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
Kindly download the complete Journal below

LETTER TO THE EDITOR

Posted onSeptember 6, 2022May 30, 2023Leave a comment

Cord Blood Banking Services in Nigeria; the Earlier the Better

Article Views: 6

LETTER TO THE EDITOR

Cord Blood Banking Services in Nigeria; the Earlier the Better

Musa Abidemi MUHIBI; PhD, FWAPCMLS, FMLSCN; Professor of Haematology and Uchejeso Mark OBETA; M Sc, MPA, AMLSCN; Assistant Director, Medical Laboratory Services

‘Haematology and Blood Transfusion Laboratory, Department of Medical Laboratory Science, Edo State University, Uzairue, Edo State, Nigeria.

Email:

Volume 1, Issue 2&3: page 180-182 | 2022. ARTICLE ID: 2022–AJLHTS–0018 www.hbtssn.org/ajlhts ISSN (Print):2814-0591; (Online): 2814-0605

African Journal of Laboratory Haematology and Transfusion Science

2Federal School of Medical Laboratory Science, Jos, Nigeria.

Corresponding

Author: Musa Abidemi Muhibi,

com

180

muhibudeen@yahoo.

Received: June 6,

2022

Accepted: August 30,

2022

Published: September

30, 2022

for the purposes

Dear Editor, Blood banking services has existed in Nigeria for ages of transfusion services. Though, there are successes achieved with the blood transfusion services, one of the major challenges is that the whole blood or blood components used cannot serve as sources of stem cells, whereas cord blood can. The unavailability of cord blood banks to provide such services creates a huge gap in the medical services thereby creating opportunities for medical tourism and capital flight.

Inherited Haemoglobin and blood disorders abound in Nigeria and have caused numerous and repeated blood transfusions among the patients. In medical care, Nigeria for example, inherited haemoglobin variant disorders include but not limited to HbS and HbC variant haemoglobin molecules as seen in Sickle cell diseases; beta thalasemias; compound beta and HbE thalasemias; and some form of non deletional alpha thalasemias. Affected individuals who are always supported by multiple regular blood transfusion may have some relief with availability of cord blood (1).

Cord blood from umbilical cord that is collected after delivery can be used as an alternative to bone marrow when transfused to haemoglobinopathy and other diseases sufferers for restoring immunological dysfunctions and for other transplantations. The cord blood stem cells, with available record, has shown that over 80 diseases. can be cured after over 50,000 transplants that were successfully done across the globe (2).

There is no doubt that haematology related diseases across the globe are equally present in Nigeria. Such diseases which can be classified into: Cancers Acute lymphocytic leukemia, Acute myelogenous leukemia, Chronic myelogenous leukemia, Myelodysplastic syndrome, Neuroblastoma, Hodgkin’s disease,

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Non–Hodgkin‘s lymphoma, and Burkitt‘s lymphoma– and Blood disorders – Sickle–cell anaemia, Fanconi‘s anaemia, Thalassemia, Evan‘s syndrome, congenital cytopenia, Aplastic anaemia, Diamond–Blackfan ana e mia, and Amegakaryocytic thrombocytopenia– abound in Nigeria. Others are congenital metabolic disorders – Adrenoleukodystrophy, Gunther‘s disease, Gaucher‘s disease, Hurler‘s syndrome, Hunter‘s syndrome, Krabbe‘s disease, Sanfilippo‘s syndrome, and Tay–Sachs‘ disease; and finally Immunodeficiencies – Adenosine deaminase deficiency, Wiskott Aldrich‘s syndrome, Duncan‘s disease, Ataxia–telangiectasia, DiGeorge‘s syndrome, Myelokathexis, Hypogammaglobulinemia and Severe Combined immunodeficiency. These diseases have been shown to have been treated with stem cells of cord blood origin (2). Haemoglobinopathies, especially sickle cell disease (SCD), is common in West Africa, Nigeria inclusive. The SCD traits have a prevalence rate of 25% and up to 30,000 children are born yearly with SCD (3).

Cord blood storage and supply should be encouraged in Nigeria, as it would be of great importance for both autologous for infants and allogeneic for others in need of the stem cells. Not minding the small quantity of blood involved and possible transmission of rare genetic disease with cord blood, it is better than bone marrow because of ease of collection, reduced samples rejection, processing time and risk of infection transmission.

Basically, the practice in Nigeria across the regions and tribes is discarding of umbilical cords by burial after delivery. This is out right wastage of good source of Haemopoietic Stem Cells (HSC) including CD34and CD‘s haematopoietic progenitor cells that could save lives more than expected. There is an urgent need to raise awareness to the public especially the reproductive ages on the need for saving the life saving products gotten from umbilical cords especially now that Muhibi et al. have discovered a safe alternative in resource–poor setting to store stem cells in a cryoprotective agents like glycerine at –20°C for up to 6 months (4). This reveals that cord blood banking is possible in Nigeria.

The effort of the National Blood Service Commission shall be incomplete without interest in Cord Blood Banking (CBB). All hands of various medical professionals, agencies and policy makers should as a matter of urgency be on deck to consider CBB as early as possible through policies and infrastructural development. The earlier the better as CBB would contribute to medical tourism and capital flight reduction and in turn strengthen Nigeria health system through good management of Haemoglobinopathy and other Diseases.

fel

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REFERENCES

Journal of Cellular Pathology, 11(1), 1–4

Angastiniotis, M. (2013). Epidemiology of Haemoglobinopathies. In: 3. Ang astiniotis M, Eleftheriou A, Galanello R; Prevention of Thalassaemias and Other Haemoglobin Disorders: Vol.1: Chapter 2; Principles [Internet]. 2nd edition. Thalassaemia International Federation, 24672827. Siva ku maran, N., Rathnayaka, I.R., Shabbir, 4. R., Wimalsinghe, S.S., Sumalimina, J.A., Jayakody, S. & Chandrasekaran, M. (2018). Umbilical Cord Blood Banking and its Therapeutic Uses.

International Journal of Scientific Research and Innovative Technology. 5(1), Olayanju, A.O., Nkanga, A.E., Olay anju, A.J., Oluwatayo, B.O., Adesina, O., Enitan, S.S. & Oladele, A.A. (2017). Cord blood banking: the prospects and challenges of implementation in Nigeria. Hematology and Transfusion International Journal, 5(4), 273–278. Muhibi, M.A., Mabayoje, V.O. & Komolafe, J.O. (2019). CD34 positive stem cells recovered from cord blood remain viable after six months of cryoprotective storage process. African

Afr J Lab Haem Trans Sci 2022, 1(2): 180 – 182

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Case Images The “Case Image” section will consider for publication short reports based on diagnostic original illustrations, in digital format. The images should illustrate an unusual or novel occurrence in the field of laboratory haematology and blood transfusion science, having a substantial learning value for readers. A description of up to 400 words should be provided alongside the image, with a maximum of five authors and at least three references; no abstract or keyword required.

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biochemical iron values of blood donors in Port Harcourt, Nigeria. Hematology. 2009; 14(6):366–70. Jacob R B, Azia M G, Christian S.G, Robinson–Mbato L, Eze E.M. Prevalence of Lewis A, Rh–c, Mand ABO/Rh–D Antigens in Bonny Kingdom Rivers State Nigeria, Journal of Medical and Dental Science Research, 2021;8(6): 15–21. Book The Institute of Medicine. A Population–based Policy and Systems Change Approach to Prevent and Control Hypertension. Washington, D.C.: The National Academies Press; 2010. Edited Book Namey E, Guest G, Thairu L, Johnson L. Data reduction techniques for large qualitative data sets. In: Guest G, MacQueen KM, eds. Handbook for Team

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Selected Inflammatory and Thrombotic Indicators Among Persons Living With HIV Infection In Southern Nigeria

Posted onSeptember 5, 2022May 30, 2023Leave a comment

Article Views: 5

ORIGINAL ARTICLE

Selected Inflammatory and Thrombotic Indicators Among Persons Living With HIV Infection In Southern Nigeria Abunimye Dennis A‘ * Akwiwu Euphoria C‘ Anyanwu Stanley O’Onukak Eme E‘ Akpotuzor Josephine O

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

Department of Histopathology and Cytology, University of Calabar, Calabar.

Abstract Introduction: Although viremia has been greatly addressed in the management of human immunodeficiency virus (HIV) infection by the advancement in antiretroviral therapy, not all HIV–associated morbidities have been resolved. Observations of increased cardiovascular risk in relation to antiretroviral therapy have been reported. Full blood count continues to be useful in disease management, and efforts are directed towards optimising its utility in medical practice. Derivatives of blood cell counts have in recent times proved to be informative with regards to the inflammatory thrombotic cycle. The utility of these derived parameters in HIV within the study locality is worth exploring.

VCC

Corresponding Author Dr Euphoria C. Akwiwu Department of Haematology and Blood

Transfusion Science, University of Calabar, Calabar Nigeria

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

Email: ecakwiwu@gmail.com

Received: May 01, 2022 Accepted: August 26, 2022 Published: September 30, 2022

Results: Significant reductions of white blood cell parameters were recorded in HIV, particularly among infected persons on antiretroviral therapy. Platelet count and plateletcrit were significantly lower, while mean platelet volume and platelet distribution width were higher in newly diagnosed persons compared to HIV–infected subjects on therapy and control subjects. Platelet–to–lymphocyte ratio was significantly higher among subjects on therapy compared to the rest of the groups.

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reasel

Conclusion: Increase in platelet count following antiretroviral therapy could be posing a risk of platelet–driven morbidities as typified in the observed elevated thrombotic marker.

Key words: HIV, immunity, antiretroviral therapy, thrombosis

V11

SIS

directed towards optimising the utility in medical practice. This is particularly in recognition of increasing burden of healthcare cost. Thus, derivable parameters that are at no additional cost to patients are gaining advocacy especially if such parameters are observed to be significantly deranged in disease states. Derivatives of blood cell counts have in recent times proved to be informative with regards to the inflammatory–thrombotic cycle (15,16). The neutrophil–to–lymphocyte ratio and platelet–to–lymphocyte ratio, in particular, are suggestively better morbidity indicators compared to the individual parameters both in health and disease conditions (17,18). The utility of these derived parameters in HIV within the study locality is yet to be explored hence the present study.

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Introduction The management of human immunodeficiency virus (HIV) infection has undergone tremendous reviews since the discovery of this medical condition. Quite early in the wake of identifying this disease entity, studies into its pathogenicity and pathophysiology brought to light the basic knowledge of its immune–related nature (1,2,3). At the forefront of the disease mechanism is the debilitating interference with

y. Hence the emergence of the CD4 cell count as a biomarker for prognosis and disease monitoring served as a major breakthrough in the management of HIV infection (4,5,6). Since then, other sensitive indicators of morbidity and overall survival such as viral load and anaemic indices have been integrated into the protocol for effective HIV management (7,8,9). Interestingly, with the advancement in HIV chemotherapy, not all HIV –associated morbidities have been addressed. It would appear that certain complications are being observed to be prevalent among infected persons on antiretroviral therapy (10,11,12,13). One such aspect of deep concern is that of HIV–associated cardiovascular involvement. Observations of increased cardiovascular risk in relation to antiretroviral therapy have been made by previous studies. More interesting is the identification of components of the full blood count, such as variations in blood cell size, as markers in this emerging field of concern(14).

The full blood count has continued to be

agement, and efforts are

Tain

LOU

Materials and methods

The present study was carried out among a total of 90 subjects at the University of Calabar Teaching Hospital at Calabar, Nigeria. Purposive sampling technique was used to enrol 30 participants each for the categories of newly diagnosed, subjects on HAART and control subjects. The subjects were between 25 and 45 years of age. Ethical approval was sought and duly obtained from the University of Calabar Teaching Hospital Health Research Ethics Committee, while informed consent was obtained from each participant.

Blood sample was appropriately obtained from each subject for w

for white cell and platelet automated estimations using Mindray BC–5000

Useru

asema

DITS are

174

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Haematology Analyzer (Mindray Medical Discussion International Limited, China). This equipment This study observed lower total and differential was controlled and calibrated according to white blood cell counts in HIV, particularly manufacturer‘s instructions to ensure its fitness among infected persons on antiretroviral for use. Blood cell ratios were mathematically therapy. Cytopenia in relation to HIV infection derived. Data generated were entered into has been among the major haematological Microsoft excel spreadsheet and analysed using derangements observed in its management. This Statistical Package for Social Sciences (SPSS) is thought to be driven by the mechanism of software version 22.0. Frequencies and one– haemato–suppression, thus suggesting the way analysis of variance were used for analysis possibility of reversal where viral load is of data. The least significant difference (LSD) sufficiently reduced (19,20,21,22,23). To this Post Hoc Test accompanied the ANOVA for end, effective administration of antiretroviral interpretation of significant variance. Statistical therapy is expected to ameliorate HIV significance was drawn at a p<0.05.

associated cytopenia in addition to arresting

viral replication and immune deficiency. Results

However, there appears to be conflicting This research on selected inflammatory and reports regarding values of peripheral white thrombotic indicators among persons living blood cell lineages following antiretroviral with HIV infection in Southern Nigeria was therapy. Decline in total white blood cell count carried out at the University of Calabar together with the granulocyte and lymphocyte Teaching Hospital, Calabar. A total of 90 male sub–populations following administration of and female subjects were enrolled in the study. antiretroviral therapy have also been reported In terms of HIV status of the participants, those by previous studies (24,25). on HAART were made up of 33.3%, those that The present study also recorded lower were newly diagnosed were 33.3%, while sero– platelet count and plateletcrit alongside higher negative control subjects were also 33.3%. mean platelet volume and platelet distribution

The total white blood cell count (WBC) width in the newly diagnosed HIV subjects. and absolute monocyte count varied The risk of thrombocytopenia has been linked significantly across the groups. Absolute to the HIV pathophysiology. Possible neutrophil and lymphocyte counts were mechanisms for its occurrence include observed to be significantly lower in HIV increasing viremia, immune response to viral subjects on HAART compared to newly invasion, disease progression and adverse effect diagnosed and control subjects (Table 1). from certain antiretroviral agents (26). There

Among the platelet parameters, platelet are reports of HIV–associated count and plateletcrit were significantly lower thrombocytopenia with a predominance of the among the newly diagnosed group compared to mild form across the African region. Findings of the other groups, while mean platelet volume these studies reveal a pattern of improved and platelet distribution width were observed platelet count following antiretroviral therapy to be higher in newly diagnosed compared to (27,28,29). In addition, lower platelet count and HIV subjects on HAART and control subjects higher platelet distribution width have been (Table 2). In addition, platelet–to–lymphocyte reported in Calabar, Nigeria (30). The study ratio was significantly higher among subjects on observed that although the finding of lower HAART compared to the rest of the groups as platelet count could arise from insufficient shown on Table 3.

production as well as increased consumption,

UT US

increas

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the finding of higher platelet distribution width value suggested the later. Platelet distribution width represents the variability in platelet size and is thought to be an important marker of platelet activation (31).

In the light of the foregoing, increase in platelet count following antiretroviral therapy without a check in platelet activation may be inimical to the management of HIV infection in the long run. Interestingly, as advancement in antiretroviral therapy continues to receive endorsement, there are also reports of associated cardiovascular disease risk (32,33,34). Several studies have linked both the infection and antiretroviral therapy with

increased risk of platelet-driven cardiovascular events, particularly myocardial infarction (35, 36, 10, 12). Thus, the finding of raised platelet to–lymphocyte ratio among subjects on antiretroviral therapy in the present study quite significant and informative as it reveals the utility of this parameter in determining the risk of cardiovascular complication. In conclusion, improvement of platelet count by antiretroviral therapy could be posing a risk of platelet–driven morbidities as typified in the observed elevated thrombotic marker.

conflict of Interest: Authors declare no conflict of interest.

Table 1. White cell parameters of study participants

Key: HAART = Highly active antiretroviral therapy, WBC = White blood cell. *Significantly different from other groups

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Table 2. Platelet parameters of study participants

Key: HAART = Highly active antiretroviral therapy, PLT = Platelet count, MPV = Mean platelet volume, PDW = Platelet distribution width, PCT = Plateletcrit. *Significantly different from other groups

Table 3. Blood cell ratios of study participants

Parameters

P–value

Subjects on HAART

Subjects newly diagnosed (n = 30)

Key: HAART = Highly active antiretroviral therapy, NLR = neutrophil–to–lymphocyte ratio, PLR = platelet–to–lymphocyte ratio Neutrophil. *Significantly different from other groups

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Association of BCL11A and HBS1L-MYB Polymorphisms in Sickle Cell Anaemia Subjects of Different Age Groups in Nigeria

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Review Article

Role of MicroRNA in Acute Myeloid Leukaemia: An Overview

Aruomaren Austin Iroghama^{1, 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

SUMMARY

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

INTRODUCTION

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.

MICRORNAS

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.

ACUTE MYELOID LEUKAEMIA

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)
RUNXI/RUNXITI t(8;21)	Core binding factor leukaemia (CBFL)	Favourable	6, 7
RUNXI/MECOM (EVI1) t(3;21)	Core binding factor leukaemia (CBFL)	Favourable	7
CBFB-MYH11 inv(16) or t(16;16)	Core binding factor leukaemia (CBFL)	Favourable	6, 7
PML-RARA t(15;17)	Acute promyelocytic leukaemia	Favourable	2, 6, 7
Trisomy 8	AML not otherwise specified	Intermediate	7, 8
Translocation t(9; 11)	Acute monocytic leukaemia	Intermediate	7
Brain and acute leukaemia cytoplasmic (BAALC) expression	Cytogenetic-normal AML (CN-AML)	Poor	2
Nucleophosmin (NPM1)	Cytogenetic-normal AML (CN-AML)	Poor	9, 10
Internal tandem duplication of fms-related tyrosine kinase gene (FLT3/ITD)	Cytogenetic-normal AML (CN-AML)	Poor	10
CCAT/enhancer binding protein alpha (C/EBPA)	Cytogenetic-normal AML (CN-AML)	Poor	1, 2
IDH1/IDH2	Cytogenetic-normal AML (CN-AML)	Poor	4, 9
c-Kit	Core binding factor leukaemia (CBFL)	Poor	3
Deletion -7q	AML not otherwise specified	Poor	7
Deletion -7	AML not otherwise specified	Poor	6, 7
Translocation t(6;9)	Acute myeloblastic leukaemia	Poor	7
ERG expression	Cytogenetic-normal AML (CN-AML)	Poor	2
Translocation t(9;22)	Philadelphia Chromosome positive-AML	Poor	7
MN1 expression	Cytogenetic-normal AML (CN-AML)	Poor	2
Deletion -5	Therapy-related AML	Poor	7
Trisomy 21	AML related to down’s syndrome	Poor	5
Cyclic-AMP responsive element binding protein (CREBBP)	Therapy-related AML	Poor	2
Trisomy 13 (Patau syndrome)	AML, minimally differentiated (M0)	Poor	5
Translocation t(1; 22)	Acute megakaryocytic leukaemia (M7)	Poor	2
Deletion -5q	Therapy-related AML	Poor	7
11q23 abnormalities	Acute monocytic leukaemia (M5)	Poor	7, 8, 11
Wilms tumor 1 (WT1)	Cytogenetic-normal AML (CN-AML)	Poor	9

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

MICRORNAs in 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	Comments	Reference (s)
FLT3	miR-16↓ miR-204↓ miR-424↓ miR-155↑	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)	miR-449A↓	Promotes apoptosis through negative regulation of NOTCH1 and BCL2	2
KIT overexpression	miR-29b↓	Forms a network with SP1/NFkB1/HDAC resulting in KIT expression	2
ERG expression	miR-196a↑ miR-196b↑	Both miRNAs regulate ERG	2
BAALC overexpression	miR-148a↓ miR-3151↑	Negatively regulates BAALC expression Targets USP40 and FBXL20 genes and is associated with poor overall survival (OS)	1, 2
CREBBP overexpression	miR-34b↓	Targets CREBBP regulating its expression	2
Erythroleukemia	miR-17↑ miR-92a↑	Targets pro-survival proteins BCL2_,STAT5, JAK2 Regulates p53 through involvement of erythroid transcription factor GATA-1	2, 8
Acute megakaryobkastic leukemia	miR-125-2↑ miR-29a↓	Reduces expression of ST18 and DICER1 Associated with poor prognosis	2, 15
NPM1 mutation	miR-29a↑ miR-20a↑ miR-15a↑ miR-10a/10b↑	Targets SERBINB9, SPARC Targets IRF2 and KIT Targets MN1,CLCN3, CRKL Anti-apoptotic; targets KLF4 and RB1CC1	1, 2, 3, 14
CEBPA mutation	miR-181a↑ miiR-34a↓ miR-223↓	Erythroid differentiation of leukemic blasts Causes E2F3 overexpression and cell proliferation Reduces expression of CEBPA suppressor NF1-A and transcription factorE2F1	1, 2
MN1	miR-16↓ miR-17-92↓ miR-126↑ miR424↑	Promotes apoptosis by targeting BCL2 Involved in malignant transformation Promotes angiogenesis Promotes macrophage/monocyte differentiation	1
t(8;21) AML1/ETO	miR-223↑ miR-126↑ miR-9↓ miR-221↓	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
t(15;17)	miR-125b↑ miR-210↓ miR-23a↓	Enhances proliferation and subdues apoptosis through regulation of pro-apoptotic protein, BAK1 Targeted by PML-RARA Targeted by PML-RARA	2
t(8;16) (p11;p13) and KAT6A-reaarangement	miR-218↑ miR-15a↑	Targets proto-oncogene RET that encodes tyrosine-kinase receptor Targets anti-apoptotic protein, BCL2	2, 6
Trisomy 8	miR-124↑	Targets myeloid transcription factor CEBPA	13
CBFL	miR-126↑ miR-221↓ miR-222↓ miR-29c↑	Same as in t(8;21) AML1/ETO Targets DNMT3A, DNMT3B and TCL1	2, 3
AML, not otherwise specified	miR-29a↓ miR-29b↓ miR-142-3p↓	Targets SP1, DNMT3A, DNMT3B, TCL1, TET1, CDK Targets CCNT2, CDK6 Targets CCNT2, CDK6	2, 21.
t(11q23) MLL-rearranged AML	miR-146a↓ miR-150↓ miR-223↑ miR-196b↑ miR-155↓ miR29a↓ miR-17-92↑ miR21↑ miR-26↑ miR-181b↓ miR-495↓	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 IN AML

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].

MICRORNA-181

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.

MICRORNA-LET-7

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.

MICRORNA-29

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.

MICRORNA-150

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.

ONCOMIRS 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).

MICRORNA-155

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.

MICRORNA-9

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].

FUTURE DIRECTIONS

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.C 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.

CONCLUSION

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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Association of Some Blood Group Phenotypes and Risk of Acute Myeloid Leukaemia In Kano, Nigeria

Posted onSeptember 3, 2022May 30, 2023Leave a comment

Article Views: 4

ORIGINAL ARTICLE

Association of Some Blood Group Phenotypes and Risk of Acute

Myeloid Leukaemia In Kano, Nigeria

*Garba, N1., Ahmad, S. G., Makuku, U.A3., and Ajayi, O.1a.

Department of Medical Laboratory Science, Faculty of Allied Health Sciences, Bayero University. P.M.B. 3011, Kano, Nigeria.

‘Department of Haematology and Blood Transfusion, Faculty of Clinical Sciences, Bayero University. P.M.B. 3011, Kano, Nigeria.

148

‘Department of Haematology and Blood Transfusion, Aminu Kano Teaching Hospital, Kano, Nigeria.

‘Department of Human Physiology, Faculty of Basic Medical Sciences, University of Benin, P.M.B.1154, Benin City, Nigeria.

Correspondence Author: ngarba.mls@buk.edu.ng,

+2348033364865

Received: April 19, 2022, Accepted: August 30, 2022 Published: September 30,

2022

Abstract

Background: Acute myeloid leukaemia (AML) is a heterogeneous clonal disorder characterized by abnormal proliferation of immature and non–functional cells known as blasts and subsequently results in bone marrow failure and organs infiltrations. Blood

group antigens have been linked with the aetiology and pathophysiology of various communicable and non–communicable diseases.

Objective: This study therefore investigated the association of some blood group phenotypes with AML among subjects in Kano, Nigeria.

Materials and Methods: Acute myeloid leukaemia was diagnosed using the clinical and cytological criteria in Aminu Kano Teaching Hospital (AKTH). The blood groups were determined using tube method for ABO, Rh–D, Lewis, Duffy and MNS while Rh–C–c–E–e and Kell by Gel method. The Antisera and Gel cards were obtained from Lorne Laboratories in the United Kingdom. All the techniques were according to the manufacturer’s instructions. Data was presented

tables and analysed using SPSS (version 25.0).

Results: A total of 25 AML subjects (age: 18.9±13.9 years, male/female ratio: 1.3:1) were studied with 25 healthy blood donors (controls) with mean ages of 29.2±5.3 years and male/female ratio: 11.5:1. Association of some blood groups and AML was determined using Chi–square test while Odds Ratio (OR) and Relative Risk (RR) were used to ascertain their risk values. The results showed that Le, Le‘, K and M antigens. were significantly associated with AML (OR: 4.75, 7.11, 11.29 and 7.67 respectively).

Afr J Lab Haem Trans Sci 2022, 1(2): 148 – 157

Garba et al / Blood Group Phenotypes and Risk of Acute Myeloid Leukaemia

Conclusion: We conclude that individuals with Lewis antigens (Le” and Le‘), Kell antigen (K) and M antigen may have increased risk of developing acute myeloid leukaemia.

Keywords: AML, Blood groups, Kano, Nigeria.

INTRODUCTION

Acute myeloid leukemia (AML) is a form of malignant disorder that is characterized by uncontrolled and exaggerated growth of immature cells known as blast cells of myeloid origin (1).

Exposure to radiation, chronic exposure to high doses of benzene, chronic heavy inhalation of tobacco smoke, some drugs used in the treatment of haematological malignancies and some genetic disorders increase the incidence of AML (2).

Acute myeloid leukemia is diagnosed on the basis of clinical and microscopic examination (cellular morphology of peripheral blood and bone morrow), immunological markers (flow cytometry), cytogenetics and molecular analysis (3). In advanced centres, immunological markers, cytogenetic and molecular analysis have reduced the important of cytochemical procedures.

Since the antigen of Red blood cell (RBC) represents inherited traits, therefore their expression should be constant throughout the life of an individual (4,5). Diminished expression of ABH antigens can occur with variant ABO alleles and in hematological disorders such as leukaemia, myeloproliferative disorders, myelodysplastic syndrome and in some cases of Hodgkin’s lymphoma. It has also been found in healthy elderly adults, pregnant females and neonates. Myeloid neoplasms have been reported to be more frequently associated with ABH antigenic alterations than lymphoid malignancies (6). Diminished expression of A

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and/or B antigens could be due to a blockage of the conversion of H substance to A and/or B substances or decreased synthesis of H antigens. Since the ABO locus encoding A and B transferases is in the 9q34 region, the chromosomal translocation (9 and 22) observed in patients with myeloid leukaemia (both chronic myeloid leukaemia and rarely AML) has also been linked to ABH red cell antigenic changes in these patients. Early recognition of the loss of ABH antigens is extremely important because it may indicate underlying sinistrous pre–leukemic states (6).

Several studies have investigated the association of blood group antigens and diseases etiology. However, no such study has been done in North–Western Nigeria. Significant results have been obtained in subjects with various diseases; For example, group A has been associated with increased risks of gallstones, colitis, and certain tumor types (7), whereas Non–O blood group

individuals have increased risk of thrombo embolic events compared to O blood groups. This is largely due to increased levels of von willebrand’s factor. These individuals have also been associated with cardiovascular diseases (8), including ischemic heart disease and atherosclerosis (9), A secretors have a higher incidence of gastric cancer (10), Non–secretors have high incidence of diseases of mouth, esophageal cancer, and epithelial dysplasia as compared to secretors (11).

This study was aimed at determining the associations of some blood group phenotypes

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Garba et al / Blood Group Phenotypes and Risk of Acute Myeloid Leukaemia

and risk of acute myeloid leukaemia in Northern Nigeria

MATERIALS AND METHODS Study Location This case–control study design was carried out at Aminu Kano Teaching Hospital (AKTH), over a period of two years (October, 2017 through October, 2019). The hospital serves as both tertiary and primary health care facility for Kano, neighbouring states and some states in the southern part of Niger Republic. Kano state is a state located in the North–western Nigeria [12]. It lies between latitude 11°30`N and longitude 8°30°E

Study Population A total of fifty (50) participants were recruited in this study, 25 were acute myeloid leukaemia patients attending AKTH and 25 apparently healthy blood donors as controls. Both males and females were recruited in this study. Ethical clearance to conduct the research was obtained from the ethics committee of Aminu Kano Teaching Hospital. Verbal informed consent for inclusion was obtained from the selected participants.

Hospital, Kano. Diagnosis of AML was made on the basis of clinical and cytological criteria (morphological and cytochemical procedures) if blasts of myeloid origin constituted at least 20% of nucleated cells in the bone marrow (3). Blood group antigenic profiles of the AML patients were determined at the teaching laboratory of the department of Medical Laboratory Science, Bayero University, Kano, Nigeria.

Three milliliters (3ml) of venous blood was collected under aseptic condition from both controls and subjects confirmed to have AML into an ethylene diamine tetra–acetic acid (EDTA) anticoagulated bottle. Samples were thereafter washed in saline before screening for blood group antigens to ABO, Rh–D, Lewis, Duffy and MNS using tube method while Rh C, Rh–c, Rh–E, Rh–e and Kell using Gel card techniques.

Five percent (5.0%) suspension of washed red cells was made in Phosphate Buffered Saline (PBS). In a labelled test tube: 1 drop of Lorne Anti–sera reagent and 1 drop of test red cell suspension were added. The tubes for ABO, Lewis, M and N antigens were incubated at room temperature while tubes for Rh–D, Duffy and S antigens were incubated at 37°C temperature for 15 minutes. The tubes were then centrifuged for 20 seconds at 1000 ref. The red cell button was gently resuspended and red macroscopically and microscopically for agglutination. AHG was added in any tube, which showed a negative or questionable result

Blood Grouping by Gel Card for Rh antigen (C,E,c,e) and Kantigen (Diamed)

Red cells from each sample was prepared as 5.0% suspension in Phosphate Buffered Saline (PBS) and Rh antigen (C, E, C, e) and K antigen typing were performed by gel card. Ten micro litre of red cell suspension was added to micro tubes followed by centrifuge at 910 rpm for 10 minutes. Agglutinated cells forming a red line on the surface of gel or dispersed in gel were

Inclusion Criteria Only subjects confirmed to have AML attending haematology day–care unit, male and female medical wards were considered eligible and recruited into this study.

Exclusion Criteria Non–acute myeloid leukaemic subjects, elderly adults, pregnant women, neonates and those who do not consent to participate in the study were excluded.

Sample Collection and Analysis Acute myeloid leukaemia was diagnosed by bone marrow aspiration at the department of haematology of Aminu Kano Teaching

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considered positive. A compact button of cells on bottom of the micro tube indicated the absence of the corresponding antigen (13).

Statistical Analysis

Data was presented as tables and figures and analysed using SPSS (version 25.0). Association of some blood groups and leukaemias were determined using Chi–square test while Odds Ratio (OR) and Relative Risk (RR) were used to ascertain their risk values. All statistical analyses were at 5% level of significance, p<0.05.

RESULTS

A total of twenty–five (25) acute myeloid leukaemic subjects and twenty–five (25) non leukaemic blood donors were studied in this research. Their ages range from 4-43 years while that of blood donors ranges from 18-40 The mean ages for AML was 18.9±13.9 years

years.

1-10

11-20

Table 1: Distribution of Acute Myeloid Leukaemia patients Based on Age Groups

Age Groups (Years)

Number of Patients (%)

13 (52.0)

3 (12.0)

2 (8.0)

5 (20.0)

2 (8.0)

25 (100)

21-30

Garba et al / Blood Group Phenotypes and Risk of Acute Myeloid Leukaemia

31-40

41-50

Total

with male/female ratio: 1.3:1 while that of blood donors was 29.2±5.3 years with male/female ratio: 11.5:1.

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AML was found to have both childhood and adulthood age distribution in this study. No AML subject was found above the age of 50 years (Table 1).

Table 2: Shows the association of blood group antigen phenotypes and risk of acute myeloid leukaemia (AML). Some blood group antigen phenotypes showed statistically significant association while some did not. The relative risk (RR) and odds ratio (OR) for all the statistically significant association were recorded. The Odds ratio given by odds of cases divided by odds of control function as Relative risk assessment that the given phenotype is associated with the leukaemia. Blood group Le“, Le‘, K and M phenotypes were significantly associated with AML (OR: 4.75, 7.11, 11.29 and 7.67).

Legend: P<0.05 indicates statistically significant difference, OR–Odds Ratio,OR<1.00 indicates protection, OR>1.00 indicates risk exposure, OR=1.00 indicates that the antigen is not a risk factor, RR=Relative Risk,D= Rhesus D antigen, C= Rhesus C antigen, c= Rhesus c antigen, E= Rhesus E antigen, e= Rhesus e antigen, Lea= Lewis A antigen, Leb= Lewis B antigen, Fya= Duffy A antigen, Fyb= Duffy B antigen, K= Kell antigen, M= MNS antigen, N= MNS antigen, S= MNS antigen.

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Ones

10U

les

DISCUSSION The clinical application of blood group antigens is mainly in the field of blood transfusion, organ donation and research purposes since the discovery of these blood group antigens. However, the use of blood group antigens in diagnosis and prognosis of various health problems is rapidly expanding as researchers had proven the association between blood group type and certain disease conditions, examples are the association between incidence of peptic ulcer and blood group O and group A with gastric carcinoma (14).

The mean ages for AML patients in this study was 18.9£13.9 years. These mean ages lesser than 44 years was reported in Jos, North Central Nigeria by Damulak et al., (15). While less than 52.1 years was reported in the South region of Nigeria by Nwannadi et al., (16). The differences in our result compared with the previous ones may be due to early development of western civilization with better health care, economic opportunities, and a resultant longer life expectancy in the Southern part of the country as postulated by Damulak et al., (15). Also, advancement in medical diagnosis as well as possible genetic predisposition may partly account for the early onset as seen in this study. However, it may no longer be true that acute leukaemias only dominates in childhood as documented earlier by Hoffbrand et al., (17). This is because AML was recorded in both children and adults in our study.

In AML, the ABO antigenic distribution is slightly deviated from the distribution seen in normal healthy non leukaemic subjects. One interesting finding was noticed on the absence of AB antigenic phenotype in AML with distribution of A antigen phenotype of 12(48.0), O phenotype of 11 (44.0) and B antigenic phenotype of 2(8.0%) while AB phenotype was 0(0%). In many other studies among blood donors, blood group O has been found to be the most common blood group and AB as least

common blood group which is slightly different from our findings where we found more A than O blood groups and no AB subjects. Eru et al., (18) reported group 52.5% and 2.2% in groups O and AB respectively amongst their participants in Benue. Looking at the distribution of ABO among leukaemic patients in our study and other studies within the country and elsewhere in the world, our finding is similar to that of Safaa (14) who found that blood group A has the highest frequency in acute non–lymphoblastic leukemia. It was also in agreement with the findings of Shirley and Desai (19) who reviewed several previously published data and found no statistically significant difference in the distribution of A blood group with respect to O blood group in patients with acute leukemias.

T he Rhesus D prevalence in this study (92.0%) is consistent with reports from previous studies among different sets of Nigerian blood donors by Olubayode et al., (20). The prevalence of Rh D was also reported by Jeremiah, (21) as 96.7%, 94% by Adeyomo and Soboye (22), 97.7% by Bakare et al., (23), 93.2% by Akhigbe et al., (24) and 93%. by Erhabor et al., (25). Safaa, (14), also showed that RhD positive blood type was detected in 91.5% of AML while Rh D negative blood type comprised only 8.5% of AML patients. The Rhesus antigens (c, C, e and E) prevalence from our study showed some dissimilarity when compared with the prevalence of Rhesus antigens in a study done previously in Kano by Baffa and Shehu (26). They reported the prevalence of the Rh phenotypes as follows e>>c>>E>>Ce (96.1%, 85.4%, 34% and 28,2%, respectively) (26).

The Lewis phenotype expression is determined by two genetic systems closely related on the short arm of chromosome 19, the Lewis genes Leand le, and the Secretor genes, Se and se. The gene product consists of various

sten

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fucosyl transferases, which from tissue liquids are adsorbed onto cell surfaces (27).

In this current study, we noticed prevalence of Le” and Le‘ as 60% and 80% respectively among AML subjects much higher than that of blood donors studied as controls. (24% and 36% respectively). We observed statistically significant associations in both Le and Le antigens with AML in this study. The Odds ratio of Le with AML was greater than 1.0. It indicated that Le positivity had greater risk than Le positivity. However, the development of these antigens after the onset of leukaemia needs to be evaluated. The critical question here is how to unravel the mechanism responsible for these predispositions.

The Duffy protein is organized on the RBC membrane as an N–glycosylated protein that spans the red cell membrane seven times (28). Fy and Fy differ by a single amino acid change at position 42 on the extracellular domain, with glycine resulting in Fy expression and aspartic acid resulting in Fy expression. Both Fy and Fy are sensitive to destruction when RBCs are treated with proteolytic enzymes such as papain or ficin (29). The prevalence of Fy” in AML subjects (24%) is significantly higher compared with that of blood donors (8%) studied (odds ratio = 3.63 and relative risk 1.66). Likewise, the distribution of Fy is higher in AML subjects (24%) while that of blood donors was (10%) (odds ratio = 7.58 and relative risk = 1.94). Isaac et al. (30), reported prevalence of Fy and Fy as 7.4% and 4.4% respectively among malaria subjects in Sokoto, Erhabor et al., (31) observed Fy and Fyb prevalence of 4.3% and 5.6% respectively among pregnant women in Sokoto as compared with the prevalence of Fy (66.0%, 99.0% and 10.0%) and Fy antigen of (83.0%, 9.2%, 23.0%) among Caucasians, Chinese and Blacks blood donors respectively.

AML had shown statistically significant association with with both Fy” (p–value

154

0.2467, OR = 3.63 and RR = 1.66) and Fy (p value = 0.0983, OR= 7.58 and RR = 1.94) antigen phenotypes in this study. Also, the mechanism involved could not be suggested in this study

The Kell antigens are located on a 93–kD RBC membrane glycoprotein that consists of 731 amino acids and spans the membrane once. The N–terminal domain is intracellular, and the large external C–terminal domain is highly folded by disulfide linkages. The Kell glycoprotein is covalently linked with another protein, called Kx, by a single disulfide bond. The Kx protein (440 amino acids and 37 kD) is predicted to span the RBC membrane ten times (32). The prevalence of Kell antigen in this study was 32% while that of blood donors studied was 4%. The Kell antigen prevalence was reported by Erhabor et al.(33) as 2% among healthy individuals in Sokoto, North Western Nigeria. Ugboma and Nwauche (34) in Port Harcourt investigated the prevalence of Kell antigen and reported a Kell antigen prevalence of 2%. Lamba et al., (35) observed a Kell antigen prevalence of 2.8% among healthy blood donors in India.

AML had statistically significant association with Kell antigen phenotype in this study (AML:p–value=0.0232, OR=11.29 and RR=2.14). This is the only blood group that recorded highest Odds ratio and serious. attention should be given to find out the mechanism of this risk exposure and the possibility of acquiring it following the onset of

AML.

The prevalence of MNS antigens in this current study among AML patients was recorded as M phenotype; 40%, N phenotypes; 92% and S phenotype; 0%. The observed prevalence from our study was at variance with the prevalence of MNS antigens among blood donors studied (M phenotype; 15%, N phenotype; 100% and S phenotype; 0%).

Makroo et al., (36) reported M prevalence

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of 88.8%, N prevalence of 65.4% and S prevalence of 54.8% among Indian blood donors.

AML had statistically significant association with M antigen phenotype in this study (AML:p–value=0.0181, OR=7.67 and RR=2.11).

CONCLUSION

We conclude that individuals with Lewis antigens (Lea and Leb), Kell (K) and M antigens have close associations with AML and may

also be predisposed further to this disease. The

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The authors want to acknowledge the assistance of the Aminu Kano Teaching Hospital, Department of Medical Laboratory Science of the Bayero University, Kano and Department of Medical Laboratory Science of the University of Benin, Benin City. Our gratitude also goes to the participants of this research.

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Isaac, I.Z., John, R.T., Udomah, F.P., Imoru, M., Erhabor, O. & Femi, A. (2016). Duffy Blood Group Distribution among Patients in a Malaria Endemic Region. International Journal of Tropical Disease and Health, 19(3), 1-5. Erhabor, O., Shehu, C.E., Alhaji, Y.B., & Yakubu, A. (2014). Duffy Red Cell Phenotypes among Pregnant Women in Sokoto, North Western Nigeria. Journal of Blood Disorders and Transfusion, 5(7), 1-5. Daniels, G. (2002). The Human Blood Groups. 2nd ed. Oxford, UK. Blackwell Science Publishing Ltd, p57-80. Erhabor, O., Malami, A.L., Isaac, Z., Yakubu, A., & Hassan, M. (2015). Distribution of Kell phenotype among pregnant women in Sokoto, North Western Nigeria. Pan African

Medical Journal, 21, 301. Ugboma, H. A, & Nwauche, C.A. (2009). Kell blood

group antigen in Port Harcourt, Nigeria–a pilot study. Port Harcourt Medical Journal, 4(2), 30-38.

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Evaluation of Iron, Ferritin levels and the Toxic effect of Datura metel in Drosophila melanogaster model.

Posted onSeptember 2, 2022May 30, 2023Leave a comment

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ORIGINAL ARTICLE

Evaluation of Iron, Ferritin levels and the Toxic effect of Datura metel in Drosophila melanogaster model. Aruomaren A. I‘, Osakue J.O‘, Ndubuisi, J. C‘. Ikponmwen, D. O‘. and Igharo O. G‘

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

ABSTRACT Introduction: The toxic effect of Datura metel have been well documented in humans however litter is known on its effect on iron profile status. The objective of this study was to evaluate the effect Datura metel on some iron profile parameters using Drosophila melanogaster model.

Corresponding Authors: Aruomaren, A. 1 Department of Medical Laboratory Sciences, University of Benin, Nigeria.

Email: iroghama.aruomaren@unib en.edu

Materials and Methods: Drosophila melanogaster flies were allocated into four classes (ethanolic leaf extract, ethanolic seed extract, aqueous seed extract and aqueous leaf extract) with six (6) different concentrations. Each concentration were performed in triplicates with twenty–five (25) flies each. Aqueous and ethanolic extract of Datura metel leaf and seed were extracted and different concentrations was administered to Drosophila melanogaster. A survival assay was done for 14 days while Serum iron and Serum ferritin assay was done after 7 days on the various treatment group. Iron and ferritin were estimated using enzyme linked immunosorbent assay (ELISA), ANOVA was used to compare between the different concentrations of the extract and the control group and result reported in mean+SEM.

Received: April 19, 2022 Accepted: August 18, 2022 Published: September 30, 2022

Results: The results obtained showed a significant decrease (p<0.001) in iron concentration in the experimental group treated with different concentrations of aqueous leaf extract of Datura metel when compared with the control. Similarly, there was a significant decrease in iron (P = 0.001) and ferritin (P <0.001) in the experimental group treated with different concentration of ethanolic extract of Datura metel seed when compared to the controls. The median survival rate

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shows that after 14 exposure to different concentrations of datura metel, aqueous and ethanolic leaves extracts had a median survival rate of 8 flies while aqueous and ethanolic seed extract had a median survival rate of 11 and 12 flies respectively.

Conclusion: The results suggest that ethanolic and aqueous extract of Datura metel seed and leaf decreases iron and ferritin levels in Drosophila melanogaster. And the leaves of Datura metel maybe more toxic than the seed of the plant.

Keywords: Datura metel, Drosophila melanogaster, iron, ferritin

INTRODUCTION Plants have always played a major role in the treatment of human traumas and diseases worldwide. The demand for medicinal plant is increasing in both developed and developing countries due to growing recognition of natural product. Herbal medicine is an important part of both traditional and modern system of medicines. For the past few years, there has been a great increase in the use of medicinal plant for curative purposes. The therapeutic activities of most plants are due to the presence of one or more of such components like alkaloids, tannins, saponins, cardiac glycosides, steroids, alkaloids, flavonoids, phenols and glycosides (1).

Datura metel is an erect shrub with spreading branches. A perennial herbaceous plant, belonging to the Solanaceae family can reach a height of 1.5m. Leaves are simple, alternate, dark green, broadly ovate, shallowly lobed and glabrous. Its common names are: Thorn apple, Devil‘s apple, Jimson weed and Angel‘s trumpet. Its indigenous names in Nigeria include: Igbo –Myaramuo; Hausa – Zakami; Yoruba – Apikan (2) Flowers are

large, solitary, and trumpet–shaped with a sweet fragrance usually appreciated in the mornings and evenings, with a wide range of colours, ranging from white to yellow and light to dark purple. The flowers are hermaphrodite and are pollinated by insects. The fruit is in the form of a capsule covered with short spines. Datura can tolerate average soil but prefers soil which is rich and moist or even very alkaline soil but hardly survives under shade. It prefers a warm temperature and is distributed in warmer regions of the world (3).

In 2016, Imo and his colleagues observed the haematological effects of ethanolic leaf, seed and fruit extracts of Datura metelin male albino rats and postulated that the decrease in RBC, PCV and Hb levels in Groups 3 administered high dose (600 mg/kg bw) of leaf extract compared with the normal control and especially 5 administered high dose (600 mg/kg bw) of seed extract is believed to be as a result of high contents of the secondary metabolites (mostly tropane alkaloids) contained in the leaf and seed of Datura metel. Other plants such as soybean have also been reported to interfere with iron homeostasis (4). Furthermore, the

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way in which Drosophila melanogaster acquires iron from the diet remains poorly understood despite iron absorption being of vital significance for larval growth (5)

Hence, this study is aimed at investigating the effects of Datura metel leaf and seed extract on serum iron and ferritin of healthy Drosophila melanogaster.

MATERIALS AND METHODS IDENTIFICATION OF PLANT The plant materials (leave and seed) were harvested in Ekosod in village, Benin City which is situated in south of Nigeria. It was further transported to the Plant Biotechnology Department, Faculty of Physical Sciences, University of Benin for authentication and given a Voucher Number UBH–321 (leaf) and 322 (seed).

Drosophila melanogaster and Treatments

Both genders (1–3 days old) of Drosophila melanogaster (Harwich stain) were cultured in the Drosophila melanogaster Research Laboratory, Department of Physiology, Faculty of Basic Medical Sciences, University of Benin, Edo state, Nigeria. The flies were obtained from the Federal University of Santa Maria, Brazil. The flies were allowed to mate in vials monitored under a regulated temperature until the eggs metamorphosed into young adult fruit flies under a natural photoperiod of about 12 hours light and 12 hours dark daily for the period of administration of the chemical compound under investigation. Flies were collected and separated into four experimental groups with five vials of 30 flies in each group and the flies were then treated as stated below in the experimental design.

PREPARATION OF FEED FOR Drosophila melanogaster

METHOD OF EXTRACTION Plant (Datura metel) extraction was carried out at the Faculty of Pharmacy, University of Benin. The leaf and seed were air dried for a period of 3 days in a cool temperate environment. The leave and seed sample was then dried after the initial time at 40°c for 2 days then it was grinded in British milling machine into powdered sample. The weight of the plant sample was recorded as 395.98g for leaf and 37.45g for seed. For Aqueous extract, the plant sample was soaked in chromatographic tank with 900ml of distilled water for 24hours, while 900ml of ethanol was used for the ethanolic extract. The liquid–soaked sample was then filtered with filter paper and funnel to differentiate the filtrate from the residue. The filtrate was then concentrated using a rotary evaporator and stored in a sample bottle for preservation in a refrigerator at 4°C.

PROCEDURE Six hundred fifty (650) ml of water was measured and turned into a pot and heated till boiling. Agar was then added to the boiling water. Yeast was dissolved using boiling water in another container. The mixture was stirred for 10 minutes. The cornmeal was dissolved with 150 ml of water with glucose added and poured into the pot and stirred continuously for 10 minutes. Yeast was then added to the mixture and stirred for another 15 minutes. The remaining 50 ml of water was used to rinse the remnant in the plate used. The meal was brought down and allowed to cool to 60°C. The preservative methylparabine was then added after it was dissolved in absolute alcohol and

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stirred. The meal was then transferred into appropriate vials.

The flies were monitored for 14 days and daily mortality records were taken.

EXPERIMENTAL DESIGN

This experiment consisted of four (4) class which were given different concentrations of ethanolic and aqueous extracts of Datura metel leaf and seed. Class 1 was (Datura metel ethanolic seed extract) Class 2 (Datura metel ethanolic leaf extract) Class 3 (Datura metel aqueous seed extract) and Class 4 (Datura metel aqueous leaf extract)

ANAESTHETIZING FLIES BY USING CO, GAS Carbon dioxide is a non–flammable odorless gas which is an excellent alternative to volatile and toxic anesthetics. It is non–toxic in low quantity. When using Co, gas, the flies are transferred to an empty vial and the CO, gas piston is shot into the vial to release the gas before they are transferred to the CO, gas panel to keep them anesthetized long enough to do each experiment.

PREPARATION OF SAMPLES FOR BIOCHEMICAL ASSAY HOMOGENIZATION The flies were collected followed by anesthetizing, and homogenising in a homogenizing buffer (0.1M phosphate buffer, pH 7.4), and centrifuged at 4000 rpm for 10 minutes. Subsequently, the supernatant was then separated from the pellet into a labelled eppendorf tubes and used for the determination of serum iron and ferritin.

In this experiment, 1800 flies were allocated into the four classes (ethanolic leaf extract, ethanolic seed extract, aqueous seed extract and aqueous leaf extract). Each of the classes were further divided into six (6) groups with different concentrations (controls, 5%

Datura metel, 10% Datura metel, 20% Datura metel, 40% Datura metel, 100% Datura metel) each. Each concentration was made in triplicates vials with twenty–five (25) flies each. The concentrations of Datura metel introduced at different concentrations into the diet in each group are presented below; Group I: Control flies fed on diet mixed with water Group II: Flies administered 5% Datura metel (ethanolic and aqueous extract) Group III: Flies administered 10% Datura metel (ethanolic and aqueous extract) Group IV: Flies administered 20% Datura metel (ethanolic and aqueous extract) Group V: Flies administered 40% Datura metel (ethanolic and aqueous extract) Group VI: Flies administered 100% Datura metel (ethanolic and aqueous extract)

BIOCHEMICAL ASSAYS Iron and ferritin were analyzed using colorimetric and Enzyme Linked Immunosorbent Assay (ELISA) methods respectively.

Determination of Iron Method: Colorimetric method Procedure Test tubes/cuvettes: “Blank“, “Standard”, “Control and “Sample” were labelled. To all tubes, 2.5 ml Iron Buffer reagent was added. To respective tubes, 0.5 ml (500 pl) of sample was added and mixed. To blank, 500 jil iron–free water was added. The spectrophotometer was

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blanked at 560 nm with the reagent blank. The was read at a wavelength of 450 nm. absorbances of all tubes were read and recorded (A1 reading). To all the tubes, 0.05 ml (50 ul) Iron color reagent was added and mixed. All RESULTS tubes were placed in the heating bath at 37°C for Toxic effect of Datura metel on Drosophila 10 minutes. The spectrophotometer was melanogaster blanked at 560 nm with the reagent blank. The To determine the effect of Datura metel on absorbances of all tubes were read and recorded Drosophila melanogaster, a survival analysis (A2 reading)

was done and to also determine which was more

potent leaves or seeds of Datura metel. Figure 1 Determination of Ferritin

panel A shows the effect of aqueous seed extract Method: Enzyme Linked Immunosorbent of Datura metel on the survival of for the period Assay (ELISA)

of 14 days. For the controls there was 41.66%

survival rate, 5% concentration showed 36.34% Procedure:

survival rate, 10% showed 27.69% survival rate, The microplates‘ well was formatted for each 20% showed 13.26% survival rate, 40% serum reference, control, and test specimen to showed 25% survival rate and 100% showed be assayed in duplicate. To each assigned 7.03%. Panel B shows the effect of the ethanol microtitre well, 0.025 mml (25 ul) of seed extract of Datura metel on the survival of appropriate serum reference, control and test Drosophila melanogaster for the period of 14 specimen was added. To each well, 0.100 ml (100 days. For the controls there was 41.66% ul) of the ferritin biotin reagent was added. The survival rate, 5% concentration showed 15.75% microplate was swirled for 20–30 secs, covered survival rate, 10% showed 15.48% survival rate, and incubated for 30 minutes at room 20% showed 17.01% survival rate, 40% temperature. The content of the microplate was showed 6.22% survival rate and 100% showed decanted and a paper was used to blot the 6.25% survival rate. Figure 2 panel shows the microplate dry. To the microplate well, 350 ul of effect of the aqueous leaf extract of Datura wash buffer was added and aspirated. This stepmetel on the survival of Drosophila was repeated thrice. To each well, 0.100 ml (100 melanogaster for the period of 14 days. For the ul) of ferritin enzyme conjugate was added. The controls, there was 41.66% survival rate, 5% content of the microplate was decanted and a concentration showed 42.42% survival rate, paper was used to blot the microplate dry. To 10% showed 36.45% survival rate, 20% shower well microplate well, 350 ul of wash buffer was 38.5% survival rate, 40% showed 33.45% added and aspirated. This step was repeated survival rate and 100% showed a 23.02% thrice To each well, 0.100 ml (100 ul) of working survival rate. Figure 2 panel B shows the effect substrate solution was added. The microplate of the ethanol extract leaf of Datura metel on well was incubated for 15 minutes at room the survival of Drosophila melanogaster for the temperature. To each well, 0.050 ml (50 pl) of period of 14 days. For the controls there was stop solution was added, the microplate was 41.66% survival rate, 5% concentration showed mixed gently for 15–20 secs. The absorbance

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35.71% survival rate, 10% showed 30.38% survival rate, 20% showed 37.43% survival rate, 40% showed 7.36% survival rate and 100% showed 7.98% survival rate. Table 1 shows the median survival rate of Drosophila melanogaster in response to the different concentrations of Datura metel. The results shows that at 100%, the aqueous seed extract showed a median survival rate of 11 flies, the ethanolic extract at 100% showed a median survival rate of 12 flies. Both the ethanolic and aqueous leaf extracts showed a median survival rate of 8 flies.

Some Iron profile parameters in Drosophila melanogaster treated with Datura metel

To evaluate the effect of the different concentrations of Datura metel on some iron profile parameters, iron and ferritin levels were estimated in both seed and leaf extracts. Figure 3 shows the effect of different concentrations (5%, 10%, 20%, 40% and 100%) of leaf extract of Datura metel on some iron profile parameters in Drosophila melanogaster for the period of 7 days. Figure 3 Panel A shows the effect of aqueous extract of Datura metel on iron. The results shows that iron was significantly lower (p=0.04) in the 100% group when compared to control. The control group

also had a significantly higher iron when compared to 40% group (p=0.003). Ferritin was also significant higher in the control group when compared to 40% (p=0.0024) administered group. 10% (p=0.016) and 20% (p=0.019) administered group also had a significantly higher ferritin levels when compared to the 40% group (figure 3 panel B). The ethanolic leaf extract of Datura metel shows that iron (p=0.0027) and ferritin (p=0.033) levels were significantly higher in the

control group when compared 100% administered group as shown in figure 3 panel C and D respectively. Figure 4 shows the effect of the seed extract of Datura metel on iron and ferritin levels in Drosophila melanogaster. Panel A shows that there was no significant difference in iron levels when comparing control to all experimental groups. However, figure 4 panel B shows that there ferritin was significantly higher (p<0.001) in the control group when compared to all experimental groups. 5% administered experimental group was significantly than 40% (p=0.001) and 100% (p=0.001) administered groups. Ferritin levels appear to be in a dose–dependent manner with the higher concentration having lower ferritin levels. Similarly, the ethanolic seed extract showed that iron levels was significantly higher (p<0.001) in the control group when compared to other experimental groups and this appear to be in a dose dependent manner (Figure 4 panel C). In the ethanolic seed extract, ferritin levels was significantly higher the control group when compared the 40% (p<0.001) and 100% (p<0.001) experimental groups. 5% experimental group was also significantly higher than 40% (p=0.003) and 100% (p<0.001) experimental groups (Figure 4 panel D).

F igure 4: Panel A and B shows the effect of different concentrations of aqueous seed extract of Datura metel on iron and ferritin levels in Drosophila melanogaster. Panel C and D shows the effect of different concentrations of ethanolic seed extract of Datura metel leaf on iron and ferritin in Drosophila melanogaster

DISCUSSION The objective of this study was to investigate the effect of aqueous and ethanolic extract of Datura metel leaf and seed on iron and ferritin

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ide dry

levels in Drosophila melanogaster. Plants have a very great potential for the treatment and management of certain disease. A large number of plants have been used by tribal and folklore: in many countries for the treatment of different diseased conditions. The medicinal value of plants lies in their bioactive phytochemical constitution that produce definite physiological actions on human body and other animals. These chemical constituents include flavonoids, alkaloids, essential oils, saponins, terpenoids, tannins and phenolic compounds. Datura metel possess potent, toxic, anticholinergic properties. The leaves are most commonly used as a narcotic, either smoked or boiled and eaten; seeds are similarly used. Roots, seeds or leaves are added to alcoholic drinks to increase the intoxicating effect. Side effects include dry mouth and throat, eye pain, blurred vision, restlessness, dizziness, arrythmia, flushing and faintness Datura species are well known because of their high concentration of tropane alkaloids, which has led to poisoning episodes when Datura is accidentally mixed with edible crops (6). In this study, results shows that the consumption of aqueous seed extract of Datura metel on the survival rate of Drosophila melanogaster for a period of 14 days, there was a moderate reduction in the survival rate of the flies (Drosophila melanogaster) on a dose dependent rate. However, there was massive reduction in the survival rate of the flies on a dose dependent rate when administered with ethanolic leaf and aqueous leaf extracts of Datura metel respectively. The median survival rate data from this study shows that the leaves of Datura metel seem to be more potent than the seed of the plant. This suggest that Datura metel may contain certain chemical that are toxic to the flies and this toxicity is increased as the dose

is increased. Although, it has been postulated that all parts of the plant are toxic because of their high tropane alkaloid level (7), the survival curve shows a massive reduction in survival rates on the ethanolic leaf extract therefore pointing to the fact that the leaves (both ethanolic and aqueous) may be more toxic than the seeds. From the survival assay, there is an indication that the toxic constituent (mostly the tropane alkaloids) of Datura metel is more soluble in ethanol than water hence a higher toxicity levels in ethanol when compared to aqueous extract. Similarly works reporting the toxic nature of datura have been reported. Fatal poisoning with Datura metel have been reported and some people have also used it as a “suicide drug” (8,9,10).

To evaluate the effect of Datura metel on some iron profile parameters, iron and ferritin were estimated. The results shows that aqueous leaf extract of Datura metel had a significant difference in the iron and ferritin concentrations among the experimental groups against the control group. There was also a significant decrease in serum iron concentration when the control group was compared to various experimental groups of different concentration. Similarly, aqueous seed extract of Datura metel had no significant effect on iron however, ferritin levels was significantly lower in the experimental groups when compared to the control group and this appear to be dose dependent. Ethanolic leaf extract of Datura metel had a significant effect on iron and ferritin concentration while ethanolic seed extract of Datura metel also significantly reduced iron and ferritin levels in the experimental groups as compared to the control group. The reduction in iron and ferritin levels is probably due to early reported observations that Datura extract

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NICI

may cause a significant disruption in hepatic and renal lipid peroxidation and this may also suggest mechanism of toxicity of D. metel in the heart which involves lipid peroxidation (11). Lipid peroxidation is a free radical–mediated oxidation of polyunsaturated fatty acids involving chain reactions that ultimately cause deleterious effect to critical biological macromolecules (12). These oxidative reactions often lead to the formation of lipid hydroperoxide which are further degraded into several aldehydic compounds including hydroxy–alkenals (13). In comparison to free radicals, these aldehydes are more stable and have the tendency to diffuse and escape from the cell thereby attacking targets cells far away. Interesting, there is a correlation between iron metabolism and lipid peroxidation which may explain our results. Similarly Okpara et al. (14) have recently reported that a significant decrease in RBC, PCV, HB as well as in the erythrocytic indices (MCV, MCH and MCHC), in albino rats administered Datura metel extract, which was attributed to RBC susceptibility to lipoperoxidative changes which is also directly association with molecular oxygen, high content of metal ions catalysing oxidative reactions and availability of high amount of polyunsaturated fatty acids (PUFA‘s). They further reported that the decrease in the erythrocyte parameters and indices in the D. metel group could also be attributed to possible disruption of haemopoisis due to intracellular oxidative stress. Haemoglobin biosynthesis takes place in the mitochondria and toxic inhibition of the activities of enzymes such as alpha aminolevulinic acid synthase (ALA–synthase) and prophobilinogen synthase will affect the haemoglobin synthetic pathway (15). The

disruption of the activities of any of these enzymes due to oxidative damage to macromolecules may interfere with haemoglobin production, thus defective and decreased erythrocyte production. Therefore, the decrease in (MCV, MCH and MCHC) values in the D. metel treated groups could also be attributed to the disruption in haeme biosynthetic pathway, hence in haemopoiesis leading to the production of microcytes. The results of iron and ferritin reported in this study could also be as result in a disruption in the heme biosynthesis pathway due to the fact that iron is a key player in the formation of heme molecule. Furthermore, the decrease in iron and ferritin levels can result in microcytic anaemia, ineffective erythropoiesis due to nutritional depletion of iron (Fe) and finally decreased haematocrit levels and haemoglobin concentration.

Most free alkaloids are lipophilic and soluble in polar solvents like methanol and ethanol (16). This may be the reason of the active effect of the ethanolic extract than the aqueous extract.

CONCLUSION Different parts of Datura metel have been reported to have therapeutic activities because of its phytochemical. Although all parts have also been reported to be toxic. The result obtained shows that ethanolic and aqueous extracts of Datura metel leaf and seed decreases iron and ferritin in experimental groups observed. Also the ethanolic extract maybe more toxic than the aqueous extract and the leaves were potent than the seeds as shown in the median survival rate.

amil

n surviva

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100

+ Controls + 5% Aqueous Seed Extract + 10% Aqueous Seed Extract

20% Aqueous Seed Extract 40% Aqueous Seed Extract 100% Aqueous Seed Extract

Survival Rate (%)

1015

DAYS

100

Controls + 5% Ethanol Seed Extract + 10% Ethanol Seed Extract

20% Ethanol Seed Extract 40% Ethanol Seed Extract 100% Ethanol Seed Extract

Survival Rate (%)

DAYS

Figure 1: Panel A shows the effect of aqueous seed extract of Datura metel on the survival of Drosophila melanogaster for the period of 14 days. Panel B shows the effect of the ethanol seed extract of Datura metel on the survival of Drosophila melanogaster for the period of 14 days.

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100

Controls + 5% Aqueous Leaf Extract + 10% Aqueous Leaf Extract + 20% Aqueous Leaf Extract + 40% Aqueous Leaf Extract

100% Aqueous Leaf Extract

Survival Rate (%)

ooo 0 0

DAYS

100

Controls + 5% Ethanol Leaf Extract + 10% Ethanol Leaf Extract + 20% Ethanol Leaf Extract

40% Ethanol Leaf Extract – 100% Ethanol Leaf Extract

Survival Rate (%)

DAYS

Figure 2: Panel A shows the effect of the aqueous leaf extract of Datura metel on the survival of Drosophila melanogaster for the period of 14 days. Panel B shows the effect of the ethanol extract leaf of Datura metel on the survival of Drosophila melanogaster for the period of 14 days.

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Table 1: Median survival rate of Drosophila melanogaster treated with different concentration of Datura metel

Aqueous Leaf Ethanol Leaf Ethanol Seed Extract Extract Extract

Concentrations Aqueous Seed

Extract Control 5% 10% 20% 40% 100%

p=0.04

p=0.003

p=0.0024

p=0.004

–0.016

2007

p=0.08

p=0.019

150

Ferritin (ng/mL)

(IP/611) ou

–H

Controls

Controls

20% Aqueous Leaf

100% Aqueous lcat

5. Aqueous leaf

10% Aqueous Leat

5% Aqueous Leaf

100% Aqueous Leaf

10% Aqueous Leaf

20% Aqueous Leaf

40% Aqueous Leaf

p=0.0027

p=0.033

Iron (vg/dl)

Ferritin (ng/mL)

Controls

5% Ethanol Leaf

20% Ethanol Leaf

40% Ethanol Leaf

Controls

5% Ethanol Leaf

20% Ethanol Leaf

40% Ethanol Lear

10% Ethanol Leaf

100% Ethanol Leaf

10% Ethanol Leaf

100% Ethanol Leaf

Figure 3: Panel A and B shows the effect of different concentrations of aqueous leaf extract of Datura metel on iron and ferritin levels in Drosophila melanogaster. Panel C and D shows the effect of different concentrations of ethanolic leaf extract of Datura metel leaf on iron and ferritin in Drosophila melanogaster.

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p<0.001

P=0.001 p=0001

p=0003

Iron (vg/dl)

Ferritin (ng/mL)

Controls

20%. Åquodas Seed

40%. Aqueous Seed $. Aqueous Seed

10% Aqueous Seed

Controls

20% Aqucous Seed

40% Aqueous Seed

100% Åqueous Seed

S%. Aqueous Seed

10% Aqueous Seed

100%. Aqueous Seed

p0.001 200.001

p<0.001

p<0.001 p=0.003

p=0.00651

200

p=0036

Iron (vg/dl)

Ferritin (ng/mL)

Controls

40% Ethanol Sood

100% Ethanol Sood‘

10% Ethanol Sood

Controls 5% Ethanol Seed

$% Ethanol Seed1

10% Ethanol Seed

20%. Ethanol Seed

20% Ethanol Seed

40% Ethanol Seed

100%. Ethanol Seed

Figure 4: Panel A and B shows the effect of different concentrations of aqueous seed extract of Datura metel on iron and ferritin levels in Drosophila melanogaster. Panel C and D shows the effect of different concentrations of ethanolic seed extract of Datura metel leaf on iron and ferritin in Drosophila melanogaster

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poisoning; identification of syncomorus and Datura Drake, L.R., Lin, S., atropine and scopolamine metel stem–bark extracts on Rayson, G.D. & Jackson, P. by high performance liquid the sperm characteristics of (1996). Chemical chromatography. Forensic Yankasa rams. Nigerian modification and metal Science International, 145, Tournal of Animal binding studies of Datura 31–39.

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Comparative analysis of different water samples as Buffer in Leishman stain.

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African Journal of Laboratory Haematology

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ORIGINAL ARTICLE

Comparative analysis of different water samples as Buffer in Leishman stain.

Okafor, Ifeyinwa Maryann, ‘Anagha Grace Olachi, Okoroiwu Henshaw Uchechi and ‘Etura, Joyce Ezekiel

Department of Haematology and Blood Transfusion Science, Faculty of Medical Laboratory Science, College of Medical Sciences, University of Calabar, Nigeria. Department of Medical Laboratory Science, Faculty of Basic Medical Sciences, Arthur Javis University, Akpabuyo, Cross River State.

Abstract Introduction Staining of blood films remains one of the most essential part of haematology. The different blood cells have different intracellular structure that take up stains according to chemical nature. Leishman stain is one of the Romanowsky stains used in the haematology laboratory for the staining of blood films. It contains methylene blue and eosin dye prepared in an alcohol medium and is diluted with a buffer. Phosphate buffer is used during Leishman stain as it acts as a mordant enhancing staining reactions and it is the recommended and best buffer for staining.

Aim This comparative study was aimed at determining if different water samples used as buffer show significant effects on the morphology of blood cells during Leishman staining.

Corresponding author: Dr. Okafor, Ifeyinwa Maryann Department of Haematology and Blood Transfusion Science, Faculty of Medical Laboratory Science, University of Calabar, PMB 1115 Calabar, Cross River State, Nigeria.

Methods pH analyses of different water sources such as water from borehole, waterboard, rainwater, fridgewater, distilled water and air conditioned water were carried out and used as buffers to carry out Leishmann staining on blood films. The stained slides were examined macroscopically and microscopically using 100x objective.

E–mail: okaforify maryann@yahoo.c om Tel: +2348080680620

Received: March 03, 2022, Accepted: July 8, 2022, Published: September 30, 2022

Results The study has shown that deionized and tap water used as buffer could produce moderately good film with well stained red cells, white cells and platelets. Rainwater, distilled, fridge and air conditioned water showed a significant change in the morphology of blood cells both in

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an apparently healthy film and a diseased film

Conclusion This study demonstrates that deionized water and tap water devoid of chlorine can be used as substitutes in the right pH of 6.8 in a situation where phosphate buffer is not available. It is therefore recommended that quality control should be carried out to check the quality of stains and buffer used for staining films in order to produce quality results. Keywords: Leishman stain, Buffer, Water

Introduction Different blood cells in our body have different shapes and sizes, which take up stains according to their structures. Some of the components of the blood cells are basophilic, that is they have great affinity to acidic dyes while some components of blood cells are acidophilic meaning they have affinity to basic dyes and also some components neutral and have high affinity to neutral dyes [1].

Romanowsky stains are types of stains employed for staining of blood cells. Almost all Romanowsky stains have two essential components; acidic and basic dye. Methylene blue is a basic dye with high affinity to the acidic component of the cell which is the nucleus, Eosin is the acidic dye that has high affinity to the basic component of the cell which is the cytoplasm and some granules of the cell [2,3]. Many Romaowsky stains are prepared with methanol which acts as a fixative and also as a cellular stain. These are four types of romanowsky stains commonly used in haematology laboratory for staining blood cells, they include: leishman stain, giemsa stain, field stain and wright stains[4,5).

Leishman stain is a type of Romanowsky stain containing a mixture of methylene blue and eosin dye prepared in an alcohol medium and diluted with buffer of pH6.8. The buffer acts as a mordant, it enhances staining reactions

and give better morphology of the blood cells under the microscope[6,7].

Leishman stain is used to stain various components of blood cells and also used to study bacterial cells. It is the basic routine stain for staining and examination of peripheral blood films under the microscope. It is also good for the examination of haemoparasite such as plasmodium species. Phosphate buffer is highly water soluble and has high buffering capacity that inhibits enzymatic activity and will precipitate in ethanol. It is one of the most used buffer employed in the haematology laboratory during Leishman staining [2,8]. Occasionally because of unavailability of phosphate buffer especially in resource–limited areas, available water samples are used as buffer.

These are different water sources that were analysed in this study: Tap water, fridge water, Distilled water, rain water and air conditioned water. Tap water is water supplied through a tap (valve) and distributed through pipes (plumbing). Fridge water is gotten from refrigerator after it has defrosted. Distilled water is steam from boiling water that has been cooled and returned to its liquid state. Rain water is collected from rainfall directly or from roof like surfaces. [3,5]. Air conditioned water is water formed from the cold coils of the air conditioner through squeezing and

in is

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Afr J Lab Haem Trans Sci 2022, 1(2): 120 – 134

Okafor et al / Comparative analysis of different water samples as buffer in Leishman Staining.

The different water sources were blindly labelled 1,2,3,4,5, and 6 for identification.

condensation of hot air in the room [4,6].

The different water types have different pH and different chemical elements in them, which may exert significant effect on the morphology of the blood cells when used in leishman staining. However, the effects of these water samples in leishman stain have not been extensively studied, hence, the reason for the study.

Materials and Methods Study Design This was a comparative and analytical study that was carried out for four months, between March to July, 2021 at University of Calabar

Teaching Hospital Calabar.

Making of blood film 1. A microscope slide was cleaned with a

cotton wool to remove debris and make it

grease free. 2. A small drop of well mixed blood sample

was placed at the centre 2cm of the length of the slide. A clean smooth edged spreader was used; the spreader is held at angle of about 45° and is drawn back to touch the drop of blood; blood is allowed to spread to the edge of the spreader. The spreader was pushed gently and quickly forward, making a film in the process; the edge of the spreader is wiped

clean. 5. The blood film was allowed to dry

completely; and slide labelled appropriately.

was

Study Area The study was carried out in Calabar Metropolis, Cross River State, Nigeria specifically at Haematology Laboratory, University of Calabar Teaching Hospital Calabar.

Sample collection

Water samples from the respective sources were collected and stored in 4 litres Jerry can and labeled accordingly as Tap water (bore hole), Tap water (water Board), Fridge water, Rain water, Air conditioner

water and Distilled water. 2. About 2ml of blood was collected from

patients with different pathological conditions (Sickle cell anaemia) and apparently healthy Individual which was used to prepare blood films.

Leishman staining 1. The air dried thin film was placed on a

staining rack with smear surface upwards. 2. The entire area of the smear was flooded

with Leishman‘s stain. 3. The flooded slide was allowed to stand for

2 minutes. To ensure cells are fixed

properly by methanol 4. The stain was double diluted with

standard buffer of pH 6.8 and also water

from other sources as buffer differently. 5. The slide was blown gently to enhance

mixture of stain with buffer; a metallic sheen is formed over the surface of the

fluid and allowed to stand for 10 minutes. 6. The stain was rinsed with tap water. 7. The reverse side of the slide was carefully

wiped with a soft absorbent tissue. 8. Slide was kept on a slide rack and allowed

to air dry.

Water Analysis Water from different sources; Tap water(bore hole), Tap water(water Board), Rain water, Fridge water, Air conditioned water and Distilled water was collected and put inside a clean container and sent to CROSS RIVER STATE WATERBOARD LTD. for analysis.

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Okafor et al / Comparative analysis of different water samples as buffer in Leishman Staining.

Constituents of phosphate buffer: 28.39g of morphology of the cells. disodium hydrogen phosphate dissolved in one PLATE 4 shows well–stained erythrocyte liter of distilled water. The pH range is checked € with faint staining of monocyte (M) and using a pH meter.

platelet (P) stained with Leishman stain using

tap water from waterboard as buffer and viewed Microscopy

with x 400 magnification. In this plate, central The stained film was air dried and viewed under pallor is not well defined and the faintly stained the microscope using x10 and x100 oil monocyte could be as a result of the amount of immersion lens. The film was chlorine present in the water used as buffer. photomicrographed using photomicrograph P LATE 5 shows well–stained erythrocyte microscope and the films were compared. €, lymphocyte (L) and platelets (P) stained using

Leishman stain using rainwater as buffer and Results

viewed with x 400 magnification. Appearance of stained blood films

PLATE 6 shows well–stained erythrocyte A well stained thin blood film appeared salmon €, neutrophil (N), and platelet (P) stained using pink.

Leishman stain using fridge water as buffer and Nucleus of the cell appeared purple–violet. viewed with x400 magnification. The Eosinophil granules appeared orange–red. erythrocytes appear crenated and not clearly Neutrophil granules appeared mauve purple. defined. Basophil granules appeared dark blue.

PLATE 7 shows erythrocyte € with Red blood cells appeared Salmon pink.

fluorescence at the center, well–stained Platelets appeared small with violet granules. neutrophil (N), and faintly–stained platelets (P) Lymphocytes and monocytes appeared pale stained using Leishman stain using distilled blue

water as buffer and viewed with x 400

magnification. The erythrocytes are not clearly PLATE 1 shows the film of an apparently defined and they appear crenated. healthy individual stained with leishman stain PLATE 8 shows moderately–stained using phosphate buffer and viewed with x 400 erythrocyte €, lymphocyte (L), and platelet (P) magnification. It shows well stained stained using Leishman stain using air erythrocytes €, eosinophil (EO), neutrophils conditioned water as buffer viewed with x 400 (N), basophils (B), monocytes (M) and magnification. The erythrocytes show crenated lymphocytes (L).

cells and are not well defined. PLATE 2 shows well–stained erythrocyte PLATE 9 shows a sickle cell film stained €, eosinophil (EO), neutrophil (N), and with leishman stain using phosphate buffer and platelets(P) stained with Leishman stain using viewed with x400 magnification. It shows well deionized water as buffer and viewed with x400 stained erythrocytes and sickle red cells. magnification. The morphology of the cells are PLATE 10 shows poorly–stained well defined without any distortion and erythrocyte €, well–stained lymphocyte (L), presence of crenated red cell.

and moderately–stained platelet (P) stained PLATE 3 shows well–stained erythrocyte using Leishman stain using deionized water as €, Lymphocyte (L), and platelets (P) stained buffer viewed with x400 magnification. The red with Leishman stain using tap water from cell morphology is distorted and appear like borehole as buffer and viewed with x400 crystals. magnification. There is no distortion in the

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Okafor et al / Comparative analysis of different water samples as buffer in Leishman Staining.

PLATE 11 shows poorly–stained erythrocyte €, moderately–stained neutrophil (N), and poorly–stained platelet (P) stained using Leishman stain using tap water from borehole as buffer and viewed with x400 magnification. The morphology of the red cells are not well defined.

PLATE 12 shows poorly–stained erythrocyte €, lymphocyte (L), and platelet (P) with numerous artefacts (A) in the background stained using Leishman stain using tap water from waterboard as buffer and viewed with x400 magnification. The red cells are not exactly well defined which could be as a result of the amount of chlorine present in the water.

PLATE 13 shows poorly–stained erythrocyte €, neutrophil (N), and platelet (P) with numerous artefacts (A) in the background stained using Leishman stain using rainwater as buffer and viewed with x400 magnification.

PLATE 14 shows poorly–stained erythrocyte € and neutrophils (N) with numerous artefacts (A) in the background stained using Leishman stain using fridge water as buffer and viewed with x400 magnification.

PLATE 15 shows poorly–stained erythrocyte €, lymphocytes (L) and neutrophil (N) with numerous artefacts (A) in the background stained using Leishman stain using distilled water as buffer and viewed with x400 magnification.

PLATE 16 shows poorly–stained erythrocyte € and lymphocytes (L) with numerous artefacts (A) in the background stained using Leishman stain using air conditioned water and viewed with x400 magnification.

Table 1 and 2 shows the result of water analysis. The table shows that different water sources has different Ph and different chemical constituents.

leishman staining by enhancing staining reaction [1]. It intensifies the stain and helps to attach the dyes methylene blue and eosin to the blood cells.

This study has shown that other water samples as buffer indeed interferes with staining results. Deionized water used as buffer was able to stain the red cells, white cells and platelets very well in a normal film as shown in plate 2, it could not stain the red cells and platelets properly but could barely stain the white cells well in sickle cell film as shown in plate 10.

Tap water from borehole used as buffer was able to stain the red cells, white cells and platelets in a normal film as shown in plate 3, it could not stain the red cells and platelets properly but moderately stained the white blood cells in a sickle cell film as shown in plate 11 whereas Tap water from waterboard used as buffer was able to stain the red cells and platelets well but was not able to stain the white blood cells properly in normal film as shown in plate 4, in sickle cell film, the red cells, white cell and platelets appeared poorly stained as shown in plate 12. The difference in staining properties that were observed in tap, deionized and waterboard water may be attributed to chlorine which is present in waterboard water but absent in both tap and deionized water.

However, Rainwater used as buffer as shown in plate 5 was able to stain the white cells, platelets and red cells very well in a normal film but in plate 13, it poorly stained the red cells, white cells and platelets in sickle cell film. Whereas Fridge water used as buffer stained the white cells and platelets very well, red cells appearing crenated as shown in plate 6 in normal film but in sickle cell film, it stained the red cells, white cells and platelets very poorly as shown in plate 14. The crenation of red cells observed in fridge water could be as a result of the slight acidic pH of the water.

Furthermore, distilled water used as buffer stained white cells well, platelets

Discussion Phosphate buffer acts as mordant during

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Okafor et al / Comparative analysis of different water samples as buffer in Leishman Staining.

appeared faintly stained and it stained the red cells poorly with the cells appearing crenated and with fluorescence at the center as shown in plate 7 in normal film while in sickle cell film, it stained very poorly the red cells, white cells and platelets as shown in plate 15.

On the other hand, air conditioned water used as buffer, moderately stained the red cells, white cells and platelets as shown in plate 8 in normal film but in sickle cell film, the red cells, white cells and platelets are poorly stained as shown in plate 16. The water sources used as buffer could not stain the cells properly because

of its inability to intensify and fix the dyes to the blood cells. Another reason the water sources were not able to stain the blood cells well could be because of their varying pH. In normal blood film, the study shows that some of the water samples such as deionized water, tap water without chlorine could be used as substitutes for phosphate buffer in a situation where it is not available or in areas where it is not easily accessible. In diseased film, the study has shown that it is best to use the recommended phosphate buffer during leishman staining for the production of better results.

TABLE1: COMPONENTS OF DIFFERENT WATER SAMPLES ANALYSED

S/N

PARAMETERS

Units

NIS

2 (Urban

10 Guideline Undergrou

Board Water)

(Rain Water)

(Fridge Drain Water)

(Distilled Water)

(AC Water)

(Deionized Water)

Borehole)

Aluminum

0.00

0.11

0.00

0.12

0.00

0.01

0.00

Ammonia NH3-N

Mg/L

0.05

0.12

0.16

0.22

1.16

0.21

0.22

Appearance

Calcium Ca

24.0

55.0

10.1

9.80

8.10

10.0

0.00

Mg/L Mg/L

50 100

0.251

2.162

0.112

0.241

0.112

0.121

0.00

Chlorine C1 Coliforms (Feacal) counts/100ml

Cfu

105

300

113

320

Coliforms (Total) counts/100ml

Colour

1.62

1.68

0.92

4.21

0.81

0.85

0.00

Copper Cu

0.12

0.11

0.15

0.16

0.15

0.00

Mg/L

2.44

2.31

3.20

3.11

3.20

3..20

0.02

Dissolved Oxygen

Electrical coductivity

148.0

504.0

88.0

24.0

0.00

Fluoride F

0.54

0.44

0.46

0.56

0.54

0.55

0.00

Mg/L Mg/L

1.0 100

10.54

34.1

0.44 68.4

17.1

12.1

Hardness (Total) as Caco,

17.1

Temperature

28.0

6.5–8.5

3.6

3.9

5.6

5.7

6.2

6.1

6.7

Ambient 6.5–8.5 200 250

– Mg/L Mg/L

28.0 3.6 6.25 8.69

2.61

6.55

2.18

1.82

1.92

pH Phosphate Total Alkalinity as CaCO, Turbidity

1.92

0.00

2.61 8.16

250

8.16

9.60

9.62

120

12.21

0.00

NTU

104

4.16

2.61

17.3

1.88

1.47

Afr J Lab Haem Trans Sci 2022, 1(2): 120 – 134

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Okafor et al / Comparative analysis of different water samples as buffer in Leishman Staining.

0,80 °•.

Plate 1– showing well–stained erythrocyte €, eosinophil (EO), neutrophil (N), monocytes (M), basophil (B), Lymphocytes (L) and platelets (P) using Phosphate

buffer. Leishman x400 magnification

Plate 2– showing well–stained erythrocyte €, eosinophil (EO), neutrophil (N), and

platelets(P) using deionized water as buffer. Leishman x400 magnification

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Okafor et al / Comparative analysis of different water samples as buffer in Leishman Staining.

Plate 3– showing well–stained erythrocyte €, Lymphocyte (L), and platelets (P) using tapwater gotten from underground borehole as buffer. Leishman x400

magnification

Plate 4– showing well–stained erythrocytet with faint staining of monocyte (M) and platelet (P) using tapwater gotten from waterboard as buffer.

Leishman x400magnification

Afr J Lab Haem Trans Sci 2022, 1(2): 120 – 134

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Okafor et al / Comparative analysis of different water samples as buffer in Leishman Staining.

Plate 5– showing well–stained erythrocyte €, lymphocyte (L) and platelets

(P) using rainwater as buffer. Leishman x400 magnification

Plate 6–showing well–stained erythrocyte €, neutrophil (N), and platelet

(P) using fridge drain water as buffer. Leishman x400 magnification

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‘Okafor et al/ Comparative analysis of different water samples as buffer in Leishman Staining.

Plate 7–showing erythrocyte € with fluorescence at the center, well–stained neutrophil (N), and faintly–stained platelets (P) using distilled water as

buffer. Leishman x400 magnification

Plate 8–showing moderately–stainederythrocyte €, lymphocyte (L), and platelet (P)

using air conditioned water as buffer Leishman x400 magnification

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Okafor et al / Comparative analysis of different water samples as buffer in Leishman Staining.

Plate 9– showing well stained erythrocytes including a sickled red cell and

platelets (P) using phosphate buffer

Plate 10–showing poorly–stained erythrocyte €, well–stained lymphocyte (L), and moderately–stained platelet (P) using deionized water as buffer.

Leishman x400 magnification

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Okafor et al / Comparative analysis of different water samples as buffer in Leishman Staining.

Plate 11–showing poorly–stained erythrocyte €, moderately–stained neutrophil (N), and poorly–stained platelet (P) using tap water from borehole as buffer. Leishman x400 magnification

erous

Plate 12– showing poorly–stained erythrocyte €, lymphocyte (L), and platelet (P) with numerous artefacts (A) in the background using tap water from water board as buffer. Leishman x400 magnification.

Afr J Lab Haem Trans Sci 2022, 1(2): 120 – 134

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Okafor et al / Comparative analysis of different water samples as buffer in Leishman Staining.

Plate 13–showing poorly–stained erythrocyte €, neutrophil (N), and platelet (P) with numerous artefacts (A) in the background using

rainwater as buffer. Leishman x400 magnification.

Plate 14–showing poorly–stained erythrocyte € and neutrophils (N) with numerous artefacts (A)

in the background using fridge water as buffer. Leishman x400 magnification.

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Okafor et al / Comparative analysis of different water samples as buffer in Leishman Staining.

Plate 15–showing poorly–stained erythrocyte €, lymphocytes (L) and neutrophil (N) with numerous artefacts (A) in the background using

distilled water as buffer. Leishman x400 magnification.

Plate 16–showing poorly–stained erythrocyte € and lymphocytes (L) with numerous artefacts (A) in the background using air conditioned water as

buffer. Leishman x400 magnification

WIG

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Okafor et al / Comparative analysis of different water samples as buffer in Leishman Staining.

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

Conclusion This study has emphasized that the type of buffer used during staining interferes with the staining procedure and affects the quality of slides produced. The study further shows that deionized and tap water from borehole could be used as substitute buffer for leishman staining in cases where the recommended buffer is not available because they are devoid of minerals such as chlorine in them.

conflict of interest: The authors declare that they have no competing interests.

Funding: There was no external source of funding.

References

1. Rana A. (2018).

Morphology of Blood Cells: Method of Preparation and Principle. Blood Biology.4(3), 3–9. Bioquest, E. (2019). Leishman Stain protocol: The Mystery Unfolds Journal of Medicine, 4(27), 4591–4606. Khayan, K.. (2019). Rainwater as a Source of Drinking Water: Health Impacts and Rainwater Treatment, 6(3), 4–9.

Bain, G. (2011). Modified Leishman Staining and effect on cell: Journal of Medicine. 4(27), 4591 4606. Bain, G. (2012). Protocol of Romanowsky staining, quality and effects, Stains, 9(2), 1–7

Wickman, F.. (2013). How Gross Is the Water That Drips From Air Conditioners? Sanitation and water safety, 6(2), 1–9. Anne, H. (2021). What is a mordant? Definition and examples. Science facts, 6(2), 3–9 Avwioro, S. A. (2011). Stud y between conventional and modified leishman stain. International Journal of Research and Review, 8(2), 5–12.

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