Leukocyte Functions During Pregnancy Amongst Nigerian Women In Edo State

LEUKOCYTE FUNCTIONS DURING PREGNANCY AMONGST NIGERIAN WOMEN IN EDO STATE

BY

ORIAZOWAN, AKHERE LIVETH

PG/BMS/1312314

DEPARTMENT OF MEDICAL LABORATORY SCIENCES

FACULTY OF BASIC MEDICAL SCIENCES

UNIVERSITY OF BENIN

BENIN CITY.

NOVEMBER, 2015

LEUKOCYTE FUNCTIONS DURING PREGNANCY AMONGST NIGERIAN WOMEN IN EDO STATE

BY

ORIAZOWAN, AKHERE LIVETH

PG/BMS/1312314

(HAEMATOLOGY AS WELL AS TRANSFUSION SCIENCE)

DEPARTMENT OF MEDICAL LABORATORY SCIENCE, FACAULTY OF BASIC MEDICAL SCIENCES, UNIVERSITY OF BENIN, BENIN CITY.

IN PARTIAL FULFILLMENT FOR THE AWARD OF MASTER OF SCIENCE DEGREE IN MEDICAL LABORATORY SCIENCE

NOVEMBER, 2015.

DECLARATION

I hereby declare that the research entitled “LEUKOCYTE FUNCTIONS DURING PREGNANCY AMONGST NIGERIAN WOMEN IN EDO STATE” is a research carried out by me under the guidance of ASSOCIATE PROF. O.I AJAYI

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ORIAZOWAN A. LIVETH DATE

CERTIFICATION

This is to certify that this research titled ‘LEUKOCYTE FUNCTIONS DURING PREGNANCY AMONGST NIGERIAN WOMEN IN EDO STATE” was carried out by ORIAZOWAN AKHERE LIVETH (MAT. NO.PG/1312314) under my supervision.

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SIGNATURE & DATE

SUPERVISOR

ASSOC. PROFESSOR. O.I. AJAYI (Ph.D., FMLSCN)

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SIGNATURE & DATE

Dr. M.A. EMOKPAE

HEAD OF DEPARTMENT,

DEPARTMENT OF MEDICAL LABORATORY SCIENCES, UNIBEN

DEDICATION

I dedicate this paper to my heavenly Father who has been my guide as well as pillar. To Him be all the glory.

ACKNOWLEDGEMENT

I appreciate most sincerely my Supervisor Associate professor, O.I. Ajayi for his tireless effort as well as immense contributions in the actualization of this research, the Assistant Dean; Dr Osadolor as well as the Head of Department; Dr. Emokpae. I want to also thank my beloved husbas well as, Rev. Eric Oriazowan for his patience, love, understas well asing as well as provisions during the course of this work. A big thank you to my lovely children Eric, Erica, Rhema as well as Praise, to my sweet mother Rev. Betty Oseghale as well as to my father, Deacon Oseghale who has gone to be with the lord, thank you for believing in me. To all my sibblings, friends as well as colleagues, thanks for your support.

May God bless you all.

TABLE OF CONTENT

Pages

Cover page – – – – – – – – – – i

Title Page – – – – – – – – – – ii

Declaration – – – – – – – – – – iii

Certification – – – – – – – – – – iv

Dedication – – – – – – – – – – v

Acknowledgement – – – – – – – – – vi

Table of content – – – – – – – – – vii

Figures as well as Tables – – – – – – – – – x

Abstract – – – – – – – – – – – xiii

CHAPTER ONE

1.1 Introduction – – – – – – – – – 1

1.2 Statement of problem – – – – – – – – 2 1.3 Justification of study- – – – – – – – – 2

1.4 Aim – – – – – – – – – – 2

1.5 Research Questions – – – – – – – – 3

1.6 Specific Objectives – – – – – – – – 3

CHAPTER TWO: LITERATURE REVIEW

2.1 What are Leukocytes – – – – – – – – 4

2.2 Pregnancy as well as some hematological Parameters – – – – – 9

2.3 Immunity – – – – – – – – – 10

2.4 Pregnancy as well as immunity – – – – – – – 12

2.5 Reactive Oxygen Species – – – – – – – 14

2.6 Leukocyte Extravasion – – – – – – – – 15

2.7 Selectins – – – – – – – – – – 18

2.8 Integrins – – – – – – – – – – 19

2.9 Cytokines – – – – – – – – – 20

2.10 Leukocytechemotaxis – – – – – – – – 21

2.11 Neutrophil migration – – – – – – – – – 23

CHAPTETR THREE: MATERIALS AS WELL AS METHODS

3.1 Ethical Consideration – – – – – – – – 27

3.2 Study Design – – – – – – – – – 27

3.3 Data Acquisition – – – – – – – – – 27

3.4 Sample size determination – – – – – – – 27

3.5 Sample Collection – – – – – – – – 29

3.6 Isolation of peripheral blood leukocytes – – – – – – 29

3.7 Full blood count analysis – – – – – – – 29

3.8 Leukocytes functional assays – – – – – – – 30

3.9 Statistical Analysis – – – – – – – – 35

CHAPTER FOUR

Results – – – – – – – – – – 36

CHAPTER FIVE

Discussions as well as Conclusion – – – – – – – – 53

REFERENCES – – – – – – – – – 57

APPENDIX – – – – – – – – – – 61

FIGURES AS WELL AS TABLES

Pages

Fig A3D pictures of Leukocytes – – – – – – – 8

Fig B showing Blood cell Lineage – – – – – – – 8

Fig C Showing the divisions of immunity – – – – – 12

Fig D micrograph showing Leukocyte migration – – – – – 17

Fig E.leukocyteExtravasion – – – – – – – – 18

Fig F . Leukocyte Chemotaxis – – – – – – – 23

Fig G. Neutrophils with segmented Nuclei – – – – – – – 24

FigH. Electron micrograph of a Neutrophil Phgocytizinganthrax bacilli – – 25

Fig I. Blood film showing NBT positive Neutrophils – – – – 25

FigJ. Blood film showing Neutrophil engulfingcas well asida – – – – – 26

Fig I Bar chart of Total Wbc count – – – – – – 41

Fig II . Bar chart of % viability – – – – – – 42

Fig III. Bar chart of % NBT- – – – – – – – 43

Fig IV. Bar chart of % phagocytosis .- – – – – – – 44

Fig V.Bar chart of % Chemotaxis – – – – – – 45

Fig VI.Correlation graph of TWBC as well as % viability – – – – 46

Fig VII. Correlation graph of TWBC as well as % NBT – – – – 47

Fig VIII. Correlation graph of TWBC as well as % phagocytosis – – – – 48

Fig IX. Correlation graph of TWBC as well as % Chemotaxis – – – 49

FigX . Correlation graph of % Chemotaxisas well as % Phagocytosis – – – 50

Fig XI. Correlation graph of% Phagocytosis as well as % NBT – – – – 51

Fig XII Correlation graph of % NBT as well as % Chemotaxis- – – – – – 52

TABLES

Pages

Table 1. Mean as well as stas well asard as well as error of mean of FBC

in control, pregnancy state,1st, 2nd&3rd trimesters- – – – – – 47

Table 2.Mean as well as stas well asard as well as error of mean of Leukocyte functional assays

in control, pregnancy state,1st, 2nd&3rd trimesters- – – – – – 48

ABSTRACT

It has been observed that women are susceptible to infections during pregnancy due to suppressed immunity. Immune suppression during pregnancy results from altered polymorphonuclear leukocyte functions. These leucocytes functions include chemotaxis, engulfing as well as intracellular killing of invading pathogens. In this study, Samples were collected by stas well asard methods from 300 patients comprising of 250 pregnant women at various gestational ages as well as 50 non pregnant women which served as control. The 250 pregnant women comprised of 50 women in their 1st trimester of gestation, 100 in their 2nd trimester of gestation, as well as 50 in their 3rd trimester of gestation.The results obtained, revealed a significantly decreased Hemoglobin concentration, Packed cell volume, lymphocyte as well as platelet counts in the pregnant women during the three trimesters when compared to the non-pregnant women who served as controls (p < 0.05, respectively). The total white cell count as well as Neutrophil count of the pregnant women increased significantly (P<0.05, respectively) when compared with values obtained from the non- pregnant women control.The leukocyte function tests which includes chemotaxis, Nitrobluetetrazolium test, as well as Phagocytic index exhibited significant decreases in the pregnant women when compared with the non-pregnant control as well as increasing ages of gestation(P<0.05, respectively). Chemotaxis was positively correlated with phagocytic index as well as Nitrobluetetrazolium (p < 0.05, respectively), while total white blood cell count did not show any significant correlation with all the functional parameters studied. We conclude that the increase in total white blood cell count with increase in gestational age coupled with decreased leukocyte functions are possible physiological responses during pregnancy. These are suggestive of depressed cellular as well as humoral immunity, the exact mechanism is however not clear.

CHAPTER ONE

1.1 INTRODUCTION

Leukocytes, also called white blood cells are immune cells that involves defending or protecting the body from both foreign invaders as well as contagious diseases. All leukocytes are produced as well as derived in the bone marrow from a multipotent cell referred to as hematopoietic stem cells. They are seen all over the body which includes the blood as well as the lymphatic system. It has been observed that white blood cells play a major role in aiding the body’s defenses against infections thereby building as well as maintaining the body’s immunity. Immunity in this way, is the adjusted condition of having satisfactory biologic defense to fight against foreign invaders or other undesirable biological intrusion, while having sufficient resilience as well as tolerance to prevent conditions such as allergy, inflammation or maladies (Maton, et al., 1997).

In pregnancy, it has been observed that there is impairment in several immunologic functions particularly maternal cell- mediated immunity, which could result in certain infections. This depression of immunity may be, to prevent the rejection of the fetus as well as also due to hormonal changes (Crouch, et al., 1995). As immunological changes build up, the equalization needed for host’s defense as well as regulation of autoimmunity is changed as well as the gravid woman acquires susceptibility to contaminations as well as changes in the disease activities hence the need to determine leukocyte function. In an early study, Persellin as well as Leibfarth, 1978, observed, that the maternal immune system has been altered. This alteration includes changes in uterine mucosa as well as the peripheral immunity.

According to Krause et al., (1987), previous studies have shown that pregnant women have a decreased neutrophil mobility (Chemotaxis) as well as it was shown that polymorphonuclear leukocyte chemotaxis was significantly decreased during the second as well as the third trimesters.

They also observed that polymorphonuclear leukocyte adherence was also significantly decreased in the third Trimester.

In view of the above, Leukocyte functions will be evaluated in pregnant women with regards to neutrophil’s chemotactic ability using the phagocytic test as well as the chemotaxis assay, as this could explain the increased incidence of infection during pregnancy as well as also in determining the immune condition of the gravid woman as well as better management during the period.

1.2 STATEMENT OF PROBLEM

In principle, chemotaxis functions as a major role in phagocytosis of invasive agents which results in the Hosts’ protection. However, this action (phagocytosis) may be impaired if the leukocytes are unable to sense as well as move toward the invading agents, thereby predisposing the individual to various infectious diseases.

1.3 JUSTIFICATION OF STUDY

Infection during pregnancy puts the mother as well as the fetus in a risky condition so accounting for high maternal as well as child’s morbidity, as well as sometimes mortality. Previous studies have shown that pregnant women have decreased leukocyte (Neutrophil) motility but results are discordant. Hence the need for this study as this will further prove if this fact is true

1.4 AIM

In this study we evaluated Leukocyte functions with regards to its chemotactic ability, as this study could explain the increased incidence of infection as well as amelioration in pregnancy hence could aid in the management of the pregnant woman throughout conception.

1.5 RESEARCH QUESTIONS

1. Are leukocyte functions impaired during pregnancy?

2. At what trimester is the leukocyte function altered?

3. What are the specific functional abnormalities of polymorphonuclear neutrophils in pregnancy?

1.6 SPECIFIC OBJECTIVES

To determine leukocyte mobility during the 1st, 2nd, as well as 3rd trimesters of the pregnant women.

To establish a relationship between Leukocyte functions as well as pregnancy

To determine the viability of phagocytic Leukocyte

To determine the phagocytic function of leukocytes in pregnancy

To determine oxidative respiratory burst of phagocytic leukocytes

CHAPTER TWO

LITERATURE REVIEW

2.1 WHAT ARE LEUKOCYTES?

Leukocytes (also known as white blood cells) are immune cells that are responsible for protecting the body from infections or foreign invaders. Leukocytes are produced in the bone marrow from the hematopoetic stem cell also called themultipotent cell. The word leukocyte directly mirrors its depiction. It is gotten from the Greek word “leuko” which means "white” as well as kytos which means "hollow vessel", as well as also -cyte is translated as "cell".

Different types of leukocytes have been identified as well as they have been differentiated by their functional as well as morphological characteristics. They include, Neutophils, Monocytes, Eosinophils, basophils as well as lymphocytes. The number of a normal leukocyte count is 4-11×109/L of blood as well as is an indication of disease. The leukocyte count underneath the lower limit is referred to as Leukopenia while the count above the upper limit is called leukocytosis (Bruce, et al., 2002).

Neutrophils commonly referred to as polymorphonuclear leukocytes constitute about 60 -70% 0f circulating leukocytes are the most numerous leukocytes, as well as are involved in the fight against microbial as well as fungal infections. Also they are multi-lobed (3-5 lobes linked by minute stras well ass) hence the name polymorphonuclear leukocytes as well as are usually the first that respond to bacterial infections. They are widely recognized cells seen in the early phases of acute inflammation as well as their life span is about 5.4 days. The cytoplasm might seem transparent as a result of smooth granules which are pale lilac in colour when they are stained. Neutrophils are dynamic in phagocytosing microbes as well as are unable to restore their lysosomes (used in degrading microbes) hence they are unable to survive after phagocytosing pathogens forming pus (Pillay, et al., 2010).

Basophils which are the fewest of the leucocytes are majorly responsible for allergies as well as antigen response. They discharge two categories of chemicals that guide in the body's barriers: histamine as well as heparin. Histamine is involved in enlarging veins as well as arteries (Vasodilation) as well as expansion the stream of the blood to the inflamed tissue. It likewise makes blood vessels more porous so neutrophils as well as clotting proteins can obtain entrance into the connective tissue all the more effortlessly. Heparin is an anticoagulant that represses blood clotting as well as advances the migration of the leukocytes into a region. They can likewise discharge chemical substances that tend to attract eosinophils as well as neutrophils to an inflamed site (Saladin, 2012).

Eosinophils which have a bi-lobed nucleus connected by a thin stras well as, contain around 2-4% of the total WBC which changes for the duration of the day, seasonally, as well as amid menstrual cycle. It elevates in light of allergies, parasitic contaminations, collagen maladies, as well as diseases of the spleen as well as CNS. Eosinophils, combats against parasitic infections as well as they are the major inflammatory cells during alleergy. They work by discharging chemicals that kills substantial parasites, for example, hook worms as well as tapeworms that are too huge for one leukocyte to phagocytize. The cytoplasm is loaded with granules that is characterized by pink-orange colouration with eosin.

Monocytes are characterized by a kidney-shaped nucleus as well as are mostly agranulated. They as well function as phagocytes like the neutrophils, but they have a longer lifespan while they play an important extra role of presenting fragments of pathogens to the T-cells to further kill them. This activity, causes an antibody reaction to be triggered. Monocytes in the long run leave the circulation as well as transform to be tissue macrophages, which scavenge dead cell wastes as well as in addition assaulting microorganisms. Neither dead cell wastes nor assaulting microorganisms can be managed viably by the neutrophils. Not at all like neutrophils, monocyte can supplant their lysosomal contents as well as are thought to have a longer viable life.

Lymphocytes are of the lymphoid origin as well as are a great deal more abundant in the lymphatics compared to the blood. The Lymphocytes are recognized by possessing a proseenly stained nucleus that might be eccentric in location, as well as a moderately little measure of cytoplasm. Lymphocytes include:

Bcells: They make antibodies that can be bounded to pathogen, initiate complement as well as improve pathogen pulverization.

T cells: includes the CD4+ T-helper cells, CD8+ cytotoxic T-cells, as well as the γδT cells.

CD4 + helper T-Cells: are T-cells showing co-receptor CD4+ referred to as CD4+ T-cells. The T-cell receptors in combination with the CD4 molecules ligate the antigenic peptide introduced on the major histocompatibility complex (MCH) class 11 molecules on antigen-presenting cells (APC). Helper T-cells produce cytokines as well as perform different roles that organize the immune system response as well as are the principal index to recognize the integrity of an individual's immunity.

CD8+ cytotoxic T-cells: These are T-cells that displays the co-receptor CD8 as well as are also known as CD8+ T cells. These cells ligate antigens exhibited on MHC 1 complex of a viral infected or tumor cells as well as destroys them. About every single nucleated cell show MHC I.

ΓδT-Lymphocytes have a different T-cells receptor (not the same as the αβ TCR seen on routine CD4+ as well as CD8+ T cells).They are seen in tissue more normally than in blood, γδ T-cells offer attributes of T-cells, cytotoxic T-cells, as well as natural Killer cells.

Natural killer cells can kill cells of the body that don't show MHC Class 1 molecules, or present stress markers, for example, MHC Class 1 polypeptide related sequence A (MIC-A). Diminished expression of MHC class I as well as up-regulation of MIC-A can happen when cells are tainted by a viral infection or get to be cancerous (Trundley as well as Muffet, 2004)

It has been observed that the first trimester of a pregnant woman is dominated by the uterine natural killer cells as well as alternatively triggered macrophages which decreases in the second trimester. It is by as well as large acknowledged that the inborn immune system is enacted amid pregnancy. The quantities of monocytes as well as granulocytes altogether increase amid pregnancy as well as these cells likewise indicate phenotypical as well as function enactment (Trundley as well as Muffet, 2004)

FIG A. Showing 3D pictures of white blood cells or leukocytes (Orkin as well as Zon, 2008)

Fig B.Showing Haematopoesis (Nathan, 2006)

PREGNANCY AS WELL AS CHANGES IN HEMATOLOGICAL

PARAMETERS

Pregnancy is a state portrayed by numerous physiological hematological changes, which might seem, by all accounts, to be pathological in the non-pregnant state. Physiological changes in pregnancy is basically affected by hormonal changes in the period. Numerous hematological changes likewise, happening amid these periods are physiological.

Amid pregnancy, the total blood volume increments by around 1.5 liters, for the most part to supply the requests of the new vascular bed as well as to make up for blood loss happening at parturition. In this regard, around 1 liter of blood is contained inside of the uterus as well as mothers blood spaces of the placenta. Increment in blood volume is, subsequently, more pronounce in multi-pregnancies as well as in iron inadequate states (Ramsay, 2010). Increment of plasma volume happens by 10–15 % at 6–12 weeks of pregnancy. This is because of an expansion in plasma renin action which proposes that the increase in the plasma volume is in light of an underfilled vascular system which results from systemic vasodilatation as well as increment in vascular capacitance, as opposed to the actual blood volume extension (Bernstein et al., 2001).The Red cell mass (driven by an expansion in the production of maternal erythropoietin) increases additionally, yet moderately less, contrasted to the expansion in plasma volume, the net result being a dunk in the concentration hemoglobin. Hence, there is dilutional anaemia. The reduction in hemoglobin is normally by 1–2 g/dL by the late second trimester as well as balances out from that point in the third trimester, when there is a lessening in maternal plasma volume (Jessica, et al., 2007).

Leukocyte counts is high during gestation with the lower reference being mostly 6,000/cumm. Leukocytosis, happening amid pregnancy is because of the physiologic anxiety impelled by the pregnant state. Neutrophils are the significant kind of leucocytes on differential counts. This is likely because of hindered neutrophilic apoptosis in pregnancy. The cytoplasm of neutrophil demonstrates toxic granules. Neutrophil chemotaxis as well as phagocytic action are discouraged, particularly because of inhibitory elements present in the serum of a pregnant woman (Chas well asra, et al., 2012).

Lymphocyte count diminishes amid pregnancy through the 1st as well as 2nd trimesters as well as expas well ass amid the third trimester. An absolute monocytosis is observed amid pregnancy, particularly in the 1st trimester, yet diminishes as pregnancy progress. Monocytes aid in avoiding fetal allograft rejection by penetrating the decidual tissue (7th to 20th week of preganacy) perhaps, through PGE2-mediated immunosuppression (Kline, et al., 2005)

Studies have demonstrated that there is decline in platelet counts amid gestation. This is referred to as "gestational thrombocytopenia." It is mostly because of hemodilution as well as somewhat because of elevated platelet activation as well as enhanced clearance. There are no complications seen in gestational thrombocytopenia with thrombocytopenia as well as children don't have serious thrombocytopenia (platelet count of ≤20,000/cum2) (shehlata, 1999).

2.3 IMMUNITY

Immunity can be referred to as the balance between having adequate biological protection to fighting against infections as well as diseases including other unneeded biological infiltration, meanwhile having high tolerance to prevent inflammation, allergy, as well as autoimmune diseases. Immunity includes both the specific, as well as the nonspecific components. The non-specific component parts functions either as a barrier or as eliminator of a wide range of pathogens despite their antigenic specificities. The immune system is made up of these components. The immune system can be divided into innate as well as adaptive components (Keller as well as Richard, 2000).

INNATE IMMUNITY

Innate immunity additionally called non-specific immunity, is the in-born resistances with which an individual is conceived. It gives resistances through a few physical, chemical as well as cellular invaders. Microorganisms initially encounters the epithelial linings, physical hindrances that line skin as well as mucous layers. Resulting general defences incorporate emitted chemical signals (cytokines), antibacterial substances, fever, as well as phagocytic reactions connected with the inflammation. The phagocytes usually express surface receptors that ligates as well as react to regular molecular patterns being expressed on invading microbial surfaces. Via these processes, the innate immune system can keep the colonization, invasion as well as spread of microorganisms (Keller as well as Richard, 2000).

ADAPTIVE IMMUNITY

Adaptive immunity involves is also known as naturally acquired immunity which usually develops when there is a unconscious or conscious contact with a pathogen, (for example, vaccination). Both the natural as well as acquired immunity are further subdivided into passive as well as active immunity. The passive immunity is gained through movement of antibodies or triggered T cells from the immune host as well as are usually short lived for only about few months—while the active immunity is incited in the host itself by antigen as well as keeps going any longer, in some cases life. A further sub-division of the adaptive immune system is portrayed by the cells included; the Humoral immune system (mediated via antibody secretion) is dynamic when the organism produces its own antibodies, as well as it is passive when the antibodies are transferred between people. Also, cellular immunity (mediated by T lymphocytes) is dynamic when the individual's own T cells are empowered as well as passive when the T cells originate from another individual (Keller as well as Richard, 2000).

FIG C. Showing the divisions of immunity(Keller as well as Richard, 2000)

2.4 PREGNANCY AS WELL AS IMMUNITY

During pregnancy, the maternal immunity is modified to actively accommodate the semi-allogeneic fetus. These modifications incorporate changes in the local immune responses, i.e., in the peripheral immune responses as well as in the uterine mucosa (Trundley as well as Moffett, 2004). The decidua is active immunologically as well as it is incorporated by huge numbers of the pregnant womans immune cells. The quantities of the diverse maternal immune cells change throughout the course of gestation. The 1st trimester is usually dominated by u-Natural Killer cells as well as alternatively by triggered macrophages. In nature, the uNK cells are immunomodulatory however are non-cytotoxic as well as together with macrophages, have been proposed to be pivotal for controlling trophoblast invasion as well as spiral artery remodeling. uNK cells as well as macrophages diminish amid the 2nd trimester. In spite of the fact that uNK cells as well as macrophages are the most abundant cells in the decidua, helper as well as cytotoxic T-cells, as well as regulatory T-cells, can likewise be seen in the decidua. They might likewise assume an imperative immunoregulatory role. Recently, dendritic cells (DCs) ) have been appeared to be seen in the decidua. The specific role played by these cells in the decidua remains unknown however, they have been seen to aid a type-2 dominant state as well as likewise take part in the induction of immunotolerance. It has been accepted generally that innate immunity is triggered during gestation. The quantities of monocytes as well as granulocytes altogether increase amid normal pregnancy as well as these cells likewise demonstrate phenotypical as well as useful actuation. Moreover, the quantity of the peripheral natural killer cells as well as their secretion of IFN-g is diminished in pregnant women as contrasted with non-pregnant women as well as it was likewise demonstrated that the proportion of NK1/NK2 cells is diminished. Such changes in NK cell populaces are essential for normal gestation, as showed when Lager et al. demonstrated that in an in vitro fertilization population no live babies were conceived when the rate of maternal peripheral NK cells was above 18%. Healthy pregnancy is usually followed by a reduction in Th1/Th2 balance as sugested by Wegmann et al. Moreover, it has been recently recognized that in spite of the fact that the Th1/Th2 balance is sacrosanct in pregnancy. The immunological paradox of gestation is so much complicated. Th17 as well as the regulatory T-cells have additionally been appeared to be included in the perplexing resistant regulation seen amid pregnancy. Different studies have as of late shown that regulatory T-cells are vital in pregnancy for enhancing immune tolerance. In peripheral circulation, regulatory T cells are numerous during early pregnancy as well as have been seen to be needed for maternal immunity to accommodate a fetal allograft (Herberts, et al., 2010). In spite of the fact that the maternal immune response fluctuates during gestation, most gravid women present a healthy gestation, recommending that the immunological changes don't drastically influence the integrity of the mother. Moreover, it has been demonstrated that pregnant women are touchier to specific contaminations such as bacterial as well as viral diseases. For example, there is an increased risk of developing clinical disease after infections such as poliovirus or hepatitis A virus during pregnancy. Additionally, it has been seen that in pregnancy increases the infectivity of cytomegalovirus, herpes simplex virus, as well as malaria (Blatt et al., 2012)

2.5. REACTIVE OXYGEN SPECIES IN PHAGOCYTIC LEUKOCYTES

When phagocytic leukocytes are suitably triggered, they use up oxygen as well as produce superoxide (02-) via a process known as respiratory burst. The respiratory burst is mediated by NADPH-oxidase complex which is a multi-protein complex that exists as a dissociated state in resting cells as well as quickly aggregates after neutrophils activation. The Neutrophil NADPH Oxide (02-) produce superoxide as well as Hydrogen peroxide(H202) following activation (Robinson, 2008). The respiratory burst includes the initiation of the protein NADPH oxidase, which delivers extensive amounts of superoxide. There is a spontaneous decay or break down of superoxide through enzymes known as superoxide dismutases (Cu/ZnSOD as well as MnSOD), to hydrogen peroxide, which is subsequently converts to hypochlorous acid (HClO) by the green heme enzyme, myeloperoxidase. It is noticed that the bactericidal characteristics of HClO can kill a microbe that was phagocytosed by a neutrophil, however, it may be a step needed for the activation of proteases.

In neutrophils, regulating the activation of NADPH oxidase is vital since the oxidase is quiescent in inactive cells but is quickly triggered after appropriate cell stimulations. Because of the toxic nature of its products, warrant the need for its high regulation as well as limited in its site of action. The activation of neutrophil Oxidase physiologically include phagocytizable particles such as microbes, yeast, as well as certain substances that triggers chemotaxis in these cells, antibodies, as well as certain bioactive lipids. f- met-Leu-phe (a chemotactic peptides) have been used as a models for studying receptor ligas well ass that is mediated by the activation of NADPH oxidase. Other non-physiological agents may also activate oxidase as well as are also vital in comprehending the regulation of phagocyte NADPH oxidase. Example of such non physiological compounds incorporates some phenol esters, retinoid, fatty acids, as well as sodium fluoride (Robinson, 2008).

2.6 LEUKOCYTE EXTRAVASION

Leukocyte extravasion also called Diapedes, involves the migration of Leukocyte from the circulation, to the areas of infection, or tissue damage. This event frames a segment of the inborn immune response, includes procurement of non-specific Leukocytes. Monocytes utilize this process without contamination or tissue harm amid their advancement into macrophages.

Leukocyte extravasion mostly occurs in the post capillary venues in which hemodynamic shear pressures are reduced. This procedure includes a few stages as chemo-attraction, moving adhersion, tight adhersion, as well as transmigration. It has been shown that leukocyte mobilization is stopped at whatever point any of these steps is stifled. The roles of leukocytes are performed for the most part in the tissues as well as these capacities incorporates phagocytosis of remote particles, creation of antibodies, as well as secretion of inflammatory response initiators (such as Histamine as well as Heparin) as well as histamine neutralization. In this way, Leukocytes assume a noteworthy part in defending an individual utilizing the blood as a transport means (Wiese, et al., 2009)

CHEMO ATTRACTION

In the presence of pathogens Leukocyte (resident macrophages) are triggered. They then secrete cytokines which includes TNFa, IL-1, as well as chemokines. TNFa, IL-1, C5a initiate the endothelial cells of the blood vessels close to the infection site to express cell adhersion molecules, which includes Selectins. It is important to note that circulating white cells are localized to the injury or infection site because of chemokines present. Chemokines belongs to a family of small cytokines secreted by cells which are capable of inducing specified chemotaxis. Chemokines have been characterized into four fundamental classes-CXC, CC, CX3C as well as XC. They apply their impact by interfacing with G-protein connected transmembrane receptors seen on the surface of their targeted cells (Melik as well as William, 2008)

ROLLING ADHESION

This occurs when circulating leukocytes ligates with their carbohydrate ligas well ass to selectin molecules that are present on the internal walls of the vessels with marginal affinity, causing leukocytes to decelerate as well as roll through the internal surface of the blood vessel walls. During this process, the transitory bonds will be formed as well as as well broken between selectins as well as their ligas well ass. For instance, the cabohydrate ligas well as for P-selectin, which is known as the P seletin glycoprotein ligas well as (PSGL-1) is communicated by different sorts of leukocytes. The coupling of PSGL-1 on the leukocyte to P-Selectin on the endothelial cells takes into account leukocyte to roll-along the endothelial surface (Maverakis, et al., 2015)

TIGHT ADHESION

The rolling of Leukocytes is triggered by macrophages by releasing chemokines which causes top integrin particles to change from the default low fondness state to a high liking state. This is helped by juxtacrine actuation of integrins by chemokines as well as soluble factors secreted by endothelial cells. In the actuated state, integrins ligates then firmly to corresponding receptors communicated in endothlial cells with high affinity. This causes the immobilization of the Leukocytes notwithstas well asing shear strengths of the progressing blood.

TRANSMIGRATION

Leukocytes usually are spread out over the endothelial cells due to the reorganization of their cytoskeletons. In this state, leukocytes can extrude its Pseudopodia as well as vent via the gaps between endothelial cells. Leukocytes transmigration usually appear as PECAM proteins, located on the surface of leukocyte as well as endothelial cell which interacts as well as proseenly pull the cell via the endothelium. Once the leukocytes have passed via the endothelium, they must pass the basement membrane. The penetration action at which is controversial, involves the proteolytic degradation of the membrane, the mechanical force, or even both. Leukocytes move to the injury or infection site in the interstitial in a chemotactic gradient. This whole event is known as diapedesis. (Sorokin, 2010).

Fig D. Micrograph showing leukocyte migration,H& E stain. (Wiese, et al., 2009)

Fig. E. Showing Leukocyte Extravasation (Muller, 2002)

2.7 SELECTINS

Selectins are substances that are quickly expressed after the activation of cytokine of endothelial cells aided by tissue macrophages. Initially, P-selectin molecules are mostly expressed by triggered endothelial cells, however within 2hrs after the activation of E-selectin expression is enhanced. Endothelial selectin ligates to cabohydrates on the transmembrane glycoproteins on leukocyte, and sialyl-LewisHYPERLINK "http://en.wikipedia.org/wiki/Sialyl_lewis_x"X.

P-Selectins: P-selectin is mainly expressed on platelets as well as endothelial cells that have been triggered as well as also its synthesis can be triggered by thrombin, histamine, compliment fragment C5a, leukotriene B4, TNFα or LPS. These cytokines triggers externalization of the Weibel-palade bodies in endothelial cells, revealing on the endothelial cell pre-formed P-selectins. P-selectin also ligates PSGL-1 as a ligas well as.

E-selectins: This is expressed on endothelial cells that is triggered as well as follows shortly after the synthesis of P-selectin, which is usually induced by cytokines including IL-1 as well as TNFα. E-selectin ligates PSGL-1 as well as ESL-1

L-selectins: L-selectins are expressed on most leukocytes, as well as are also known to ligate MadCAM-1, GlyCAM-1, as well as CD34 as ligas well ass.

The impeded results of most selectins result in a reduced immune response. When L-selectin is not synthesized, immune response may be ten times reduced as P-selectins ligate to themselves. P-selectins can ligate themselves with high affinity, however occur less often due to the density of the receptor-site is reduced as compared to the small E-selectin . This enhances the first WBC rolling-speed, promoting the reduced rolling phase (MCEver, et al., 1989).

2.8 INTEGRINS

Integrins that are basically expressed on leukocytes β2 integrins are mostly involved in cell adhesion as well as are seen on rolling leukocytes which helps to ligate endothelial cell adhersion molecules, thus preventing cell movement.

LFA -1 are seen on circulating leukocytes, as well as ligate ICAM-1 as well as ICAM-2 located on endothelial cells.

MAC -1 are seen on circulating leukocytes, as well as ligates ICAM-1 located on endothelial cells.

VLA -4 are seen on both leukocytes as well as endothelial cells, as well as enhances chemotaxis. It as well ligates VCAM -1.

The cellular activation through extracellular chemokines leads to pre-formed β2 integrins to be secreted from cell stores. Integrin molecules moves towards the surface of the cell as well as then aggregate in high-avidity patches. Leukocyte cytoskeleton associate with Intracellular integrin domains via cytosolic factors mediated such as α-actinin, talin, as well as vinculin. This interaction leads to a conformational shift in the tertiary structure of integrin thus allowing ligas well as to access the ligating site. Divalent ctions (e.g. MgHYPERLINK "http://en.wikipedia.org/wiki/Magnesium"2HYPERLINK "http://en.wikipedia.org/wiki/Magnesium"+) are needed for integrin-ligas well as ligating (Aplin, 1998).

ICAM-1 as well as VCAM-1 are triggered by inflammatory cytokines, whereas ICAM-2 is jointly expressed by some endothelial cells but down-regulated via inflammatory cytokines.

Amid chemotaxis, cell migration is enhanced by the ligating of β1 integrins to the components of the extracellular matrix: VLA-4, VLA-3, as well as VLA-5 to fibronectin as well as VLA-2 as well as VLA-3 to collagen as well as other extracellular matrix components (Aplin, 1998).

2.9. CYTOKINES

Extravasation is maintained by the basic cytokine matrix synthesized mainly by the inflammatory response, as well as is not dependent on specific cell antigens. Vasodilation as well as lower electrical charge is induced by cytokines that are released in the first immune response triggered along the surface of the vessel. The flow of blood is lowered, thus enhancing intermolecular ligating. IL-1 initiates vascular endothelia, resident lymphocytes, as well as TNFα enhances the permeability of blood vessels as well as activating vascular endothelia. CXCL8 (IL-8) produces a chemo-tactic gradient which directs the leukocytes to the area of tissue injury or infection. CCL2 possess a same role as CXCL8 which induces the extravasation of monocyte as well as transforming it to macrophages as well as also activates leukocyte integrins.

2.10 LEUKOCYTE CHEMOTAXIS

This is the reaction of white blood cells to substances produced during reactions that is immunologic, where leukocytes tend to be attracted to as well as aggregate at the area of the reaction. It is a cellular function, as well as involves the attraction of inflammatory cells mostly those of monocytes as well as neutrophils, whose phagocytic activity is due to chemical influence produced by the invading organism that includes foreign substances produced by the microbes as well as endogenous acute inflammatory reacting substances (clotting factors, histamine, complement factors, kinins, platelet-activating factor, leukotrienes, cytokines, as well as prostaglas well asins). Positively chemotactic movement occurs towards the higher chemical concentration, while negatively chemotactic movement occurs when it is oppositely oriented in its direction. Chemokinesis is when a movement is chemically initiated.

One of the most widely used cell types for studying human cell chemotaxis are the blood leukocytes. These cells are white blood cells which are main ingredients in inflammatory reactions as well as the main inflammation mediators, where monocyte/macrophages as well as neutrophils serves as the major effectors of both acute as well as chronic inflammation. In responding to an inflammatory signals, circulating leukocytes binds to as well as crossing the endothelium of blood vessels as well as retreating home to most tissue sites or join in inflammatory response in the vascular walls, which depends on the nature and location of the specific tissue injury or stimulus. The movement of white cells from the circulation to the tissues is propelled by some chemotactic factors known as chemokines, which are a group cytokines of small molecular weight that function to attract cells in a gradient-dependent manner. These viable molecules are ligands for a G-protein-coupled transmembrane receptors (Cathcart1, 2009).

In exception to the skin, Neutroplis are usually the first-line of defense towards microbial infections. Neutrophils are the main phagocytes usually seen in the circulation. At the initial (acute) phase of inflammatory reaction that is mostly due to microbial infection, environmental exposure, as well as some cancers. Neutrophils are among the initiators of inflammatory cells to move to the site of the inflammation. They move via the vessels, then via interstitial spaces with the aid of chemotaxis triggered by chemicals such as C5a, FMLP (-Formyl-methionine –leucine- Phenyl alaninn), Interleukin-8 (IL-8), as well as Leukotriene B4 (Waugh and Wilson, 2008).

The Movement of cells was initially detected from the beginning of microscopy where it was described as chemotaxis by T.W. Engelmann in the year 1881 as well as W.F. Pfeffer (1884) in bacteria and H.S. Jennings (1906) in his work, ciliates.

White blood cells are mobilized from the blood to the tissue sites of the inflammation by series of various processes that takes place according to the following sequence: rolling, activation, firm adhesion, extravasation as well as chemotaxis. The first reactions that occurs between white blood cells and the endothelium are transient low-affinity adhesions which are aided by endothelial selectins as well as their ligands expressed by the cells. This results in rolling of the white blood cells along the surface of the endothelium. The rolling on the endothelium by the leukocytes interact chemo-attractants that binds specific receptors on the leukocyte, which triggers intracellular signals that initiates leukocyte’s integrins. Triggered integrins then aids high-affinity adhesive interactions between the white blood cells as well as the endothelium, by ligating endothelial intercellular adhesion molecules (ICAMs), and thus results in strong adhesion as well as arrest of rolling leukocytes. After the arrest, leukocytes migrates through the endothelium towards the tissue to sites of inflammation.

Fig F.Showing LeukocyteChemotaxis (Luster as well as Tager, 2004)

2.11 NEUTROPHIL MIGRATION INTO INFLAMMATORY REGIONS

Within the micro vasculature neutrophils moves passively in the blood circulation. The Neutrophil granulocytes move from the vessels towards the matrix, releasing proteolytic enzymes, so as to degrade intercellular linkages as well as to engulf bacteria via phagocytosis. Neutrophils usually undergo chemotaxis that enables them to move to the sites of inflammation or infection. Neutrophils are allowed to detect chemical differences between molecules by surface receptors which include interleukin-8 (IL-8), C3a, C5a, interferon gamma (IFN-gamma), as well as Leukotriene B4. They possess numerous specific receptors, which includes the complement receptors, cytokine receptors for the interleukins as well as gamma interferon (IFN-gamma), receptors to detect as well as adhere to endothelium, chemokines, lectins receptors, proteins, Fc receptors for opsonin. At the areas proximal to inflammation, the endothelial cells change their charge in response to interleukins such as IL-8, so that neutrophils can stick. Neutrophils move along the dimensional surface of the vessel as well as then squeeze between the endothelial cells. Neutrophils then invade the 3- Dimensional space as well as begin to detect the FMLP (formyl- methionine- Leucine-phenylalanine) as well as C5a gradient same time as a negatively charged interleukin-8, LTP4 gradient (Nathan, 2006).

Fig G. showing segmented Neutrophils nuclei that is surrounded by red blood cells where intra-cellular granules can be seen in the cytoplasm (Giemsa stain) (Nathan, 2006).

Fig H: showing scanning an electron micrograph of a neutrophil (yellow) that has

phagocytose anthrax bacilli (orange) (Hickey as well as Kubes , 2009).

Fig I. Bloodfilm showing NBT positive neutrophils (Robinson, 2008)

Fig J . Blood film showing Neutrophils engulfing cas well asida

(Grisafi as well as Fink, 2007)

CHAPTER THREE

3.0. MATERIALS AND METHODS

This study was carried out in order determine Leukocyte functions during pregnancy using the Leukocyte Viability test, Nitroblue tetrazolium, phagocytosis as well as chemotaxis assays . Full blood count was also carried out. These assays were carried out on pregnant women in their various gestational ages as well as non-pregnant women served as controls.

3.1 ETHICAL CONSIDERATION

This study was carried out upon the ethical approval of the committee of the Edo state ministry of Health, Benin City. Also, written informed consents were gotten from the patients.

3.2 STUDY DESIGN

This research is being carried out on pregnant women during the three trimesters of pregnancy, who were attending the ante-natal clinic at the Stella Obasanjo hospital in Benin city as well as also non pregnant women who are apparently healthy served as control.

3.3 DATA ACQUISITION

Patients were registered as well as taken through the routine ante-natal booking procedures which included getting their personal data( name, age, address, occupation, date of last menstrual period, expected date of delivery, etc),

3.4 SAMPLE SIZE DETERMINATION

The sample size for this study was 300 women (ages 18-45, of different ethnic groups in Edo state) which included 250 pregnant women while the remaining 50 apparently healthy women of child bearing age served as control. This size was derived based on 3 factors:

The prevalence estimation of interest

Confidence interval of 95%

The acceptable margin of error(0.05)

The sample size was then calculated via the formula:

N = t 2x p (1-p)

M2

or N= z2pq

M2

N = required sample size

t = Confidence level 95 % (Standard value of 1.96)

m2 = 3.8416

P = Estimated prevalence of pregnant women=0.26

M = Margin of error at 5% (Standard value = 0.05) as well as m2 is 0.0025

q = 1-p, that is 1- 0.26= 0.74

Therefore,

N = 3.8416 x0.26(0.74)

0.0025

N= 296.65 = 300

3.5 SAMPLE COLLECTION

About 8mls of blood sample was collected. 5mls in a sodium heparinized tube as well as 3mls in pottassiumEDTA( Ethylene diamine Tetra acetic acid) by stas well asardized venous collection technique. It was gently mixed to avoid clotting.

3.6 ISOLATION OF PERIPHERAL BLOOD LEUKOCYTES

Cells were isolated using lymphocyte seperating medium(LSM) (sigma, Bristol Scientific co. Nigeria) (Akhtaret al., 2010). Cells at the inerphase were washed with Hanks Balanced Salt solution(HBSS) as well as finally resuspended in Dulbecco ‘s modified Eagle medium (sigma, Bristol Scientific co. Nigeria) as well as the seeding count was done.

3.7 FULL BLOOD COUNT ANALYSIS

The Full blood count was done using the Sysmex Automated hematological

Analyser KX -21N ( Sysmex corporation, kobe Japan, 2006. code No. 461 – 2265 -5).

Principle

Sysmex KX -21N uses 3 detector blocks as well as two kinds of reagents (cell pack, Stromatolyser –WH, as well as replenishing reagent) to analyse the Blood. The WBC (White blood cell count) is performed by the WBC Detector utilizing the DC (Direct Current) Detection method. The RBC (Red Blood Cell) count as well as platelets are taken by the RBC Detector block, also using the DC Detection technique. The Hemoglobin (H B) Detector block measures the HB concentration using the Non-cyanide using Hemoglobin method.

Protocol

The blood samples were collected in EDTA containers (Ethylene-Diamine-tetra acetic acid) are mixed properly.

The stopper of the container is opened as well as set to the sample probe .

The start switch is pressed gently with the tube still under the probe.

The tube will be held under the sample probe a beep is heard with the LCD

Screen displaying “ Analysing”. Then the tube is gently removed from the Sample probe.

The unit uses automatic analysis as well as displaying the result on the LCD screen which is then printed out.(sysmex corporation, Kobe, 2006)

3.8 LEUKOCYTES FUNCTIONAL ASSAYS

Below are the various test which were used to assess leukocyte functions;

VIABILITY TEST (TRYPAN BLUE TEST)

This is also called dye Exclusion test for viable cell counting. It involves the use of Trypan Blue, product no T8154, (H7901). Trypan blue has a higher affinity for serum proteins more than for cellular protein.

Principle

The reactivity of trypanblue (sigma, Bristol Scientific co Lagos, Nigeria) is due to the fact that the chromophore is negatively charged as well as does not interact with the cell unless the membrane is damaged. Hence, all cells which are excluded from the dye will be viable.

Protocol

1. 0.5ml of the suitable Leukocyte cell suspension (diluted cells in a balanced salt solution, HBSS product no. H2513) without serum with an approximated concentration of 1 x 105 – 2 x105 cells /ml) was placed in a screw cap test tube.

2. 0.5ml of 0.4% trypan blue solution (Sigma Aldrich) was transfered to the tube containing the cell suspension

3. It was thoroughly mixed as well as allowed to stand for 5min at 15-30 0c (at room temperature).

4. The trypan Blue stain /cell suspension mixture was carefully placed into a haemocytometer with coverslip in place for cell counting.

5. Using a microscope of objective x10, cells were observed that picked up the trypan blue stain (non viable) as well as those which did not (viable) were counted as well as % was obtained using the formula;

Total viable cells (cells that excluded the dye) ÷ Total cells ( viable as well as non viable) x 100 . (Freshney, 1987)

PHAGOCYTOSIS FUNCTION TEST

Principle

It measures the percentage of neutrophils or monocytes that have ingested microbes as well as their activity(number of bacteria/fungi per cell) (Noah et al., 1995).

Protocol; Based on modified version of the method introduced by Lehrer as well as Cline (Noah et al., 1995)

1. 100ul of Leukocyte suspension at an optimum concentration of 1×106 cell/ml was mixed with 100ul (0.1ml) of E. Coli ( 3 x 10 8 cells / ml) suspension in the presence of 40 % serum

2. The mixture was incubated at 37 o c in a water bath for 1 hour.

3. After incubation, cells were fixed in 2.5% glutaraldehyde, stained with methylene Blue

4. It was then examined microscopically using the x40 objective for dead as well as live E coli within the leukocytes.

5. 200 cells were counted (including both viable cells that excluded the dye as well as dead E coli- blue stained).

NITROBLUE TETRAZOLIUM TEST (NBT)

When phagocytic leukocyte are well stimulated they consume more oxygen as well as produce super oxide (02). This process often called Oxidative respiratory burst (Robinson, 2008).

Principle

Leukocytes ingest the dye, Nitrobluetetrazolium (prepared by dissolving 200mg of NBT in 100 cc of phosphate Buffered saline solution.) (Sigma, Bristol co. Lagos, Nigeria), as well as when a reactive oxygen species is present, the yellow-coloured NBT is changed to purple-blue formazan compound (Abeer, 2013).

Protocol

Approximately 0.1ml of Heparinized blood with as well as without stimulant (Endotoxin from Ecoli product no. 0157.H7) were transferred into plastic tubes as well as mixed with an equal amount of 0.2% NBT solution (prepared by dissolving 200mg of NBT in 100cc of phosphate buffered saline solution).

The mixture was incubated at 37oc for 15 minutes in a water bath. As well as subsequently kept at room temperature for additional 15minutes.

After incubation, the solution was mixed gently as well as smears were prepared .when dried, they were stained with Leislhman stain as a counter stain

Smears were counted microscopically as well as only the neutrophils with large blue black formazan deposits were classified as NBT positive Neutrophils.

The neutrophils containing formazan deposits were counted as well as reported as a percentage.

CHEMOTAXIS ASSAY USING THE MODIFIED BOYDEN CHAMBER

Chemotaxis (with the Boyden chamber technique) as well as N- formyl –leucine – methionine- Phenylalanine (flmp) as achemotactic factor was used to evaluate Leukocyte function

Principle

Quantitation of chemotaxis in a Boyden chamber embodiment relies on the number of cells that have migrated from the top of the membrane as well as fall through the micro –pore of the track, to the bottom collection chamber , typically a reservoir microplate(Schroeder as well as Bradley, 2014)

Cells which are attracted to the chemoattractant tend to migrate towards the chemoattractant N- formyl_ met_ Leu_phe(Sigma, Bristol Scientific co. Lagos, Nigeria). The distance travelled (in microns) by the leading front of cells used to measure the chemotactic index (CI) during incubation

Protocol

1 .Migaration chambers were put in 24 well plates (Lower Chamber)

2. 100ul serum free medium was added in chamber (Upper Chamber) as well as 200ul of cell (2.5 x10 9/ml) in the serum free medium.

3. 750ul culture medium (with serum) the lower chamber containing chemo attractant

4. It was incubated at 37 oC for 12 – 16 hours

5. Medium from the chamber was removed, washed twice in PBS ph 7.2

6. Cells were then fixed by formalin (3.7% in PBS) as well as then at room temperature for 2minutes.

7. Formaldehyde was removed as well as cells washed twice in PBS

8. Cells were permeabilized by absolute Methanol as well as then at room Temperature for 20minutes.Methanol was removed as well as cells washed 2 times in PBS.

9 .Cells were then stained in Giemsa for 15 minutes .After which Giemsa was removed as well as washed twice in PBS.

10. Non migrated cells were scraped off using cotton swabs while migrated cells were counted under a light microscope. (Shaiegan, et al., 2002)

3.8. STATISTICAL ANALYSIS

Data obtained were entered in to SPSS Statistical package version 22.0 as well as statistical analysis was done using one way single factor anova. Results obtained were represnted as tables as well as figures. A significant value was set at p < 0.05

CHAPTER FOUR

RESULTS

Data obtained were from the 300 women from the ages of 18yrs to 45yrs, from different ethnic groups in Edo State. They comprised of 50 pregnant women(1st Trimester), 100in their 2nd trimester, as well as 100 in their 3rd trimester, while 50 non pregnant women served as control

Table 1: shows that the Full blood count values of the pregnant women varied from the non pregnant women as the packed cell volume, hemoglobin concentration, as well as platelet count of the pregnant women revealed a significant reduction in the values obtained (p <0.05) .The Total white cell count as well as diferential white cell count(neutrophils as well as monocytes) showed a significant increase when compared to the non-pregnant women.( p< 0.05) .

Table 2: Shows the Leukocyte function tests (%phagocytosis, % NBT (Nitrobluetetrazolium test) as well as % chemotaxis ) showed a significant decrease when compared to the non pregnant women as p < 0.05 . Dye exclusion test for leukocyte viability showed no significant difference for both the non pregnant women as well as the pregnant women in their various gestational periods ( P > 0.05).

Fig I: Bar chart comparing control, pregnant state, 1st , 2nd as well as 3rd trimester means of Total white blood cell count shows significant increases when compared to the non pregnant control (p< 0.05)

Fig II: Bar chart comparing control, pregnant state, 1st , 2nd as well as 3rd trimester means of percentage viability shows that there wasn’t significant changes in the percentage vibility of both non pregnant as well as pregnant women in their various gestational ages(p< 0.05)

Fig III: Bar chart comparing control, pregnant state, 1st , 2nd as well as 3rd trimester means of percentage Nitrobluetetrazolium. shows that the Total white cell count of the pregnant women was significantly decreased when compared to the non pregnant control (p< 0.05)

Fig IV: Bar chart comparing control, pregnant state, 1st , 2nd as well as 3rd trimester means of percentage phagocytosis showssignificant decreases when compared to the non pregnant control.(p<0.05).

Fig V: Bar chart comparing control, pregnant state, 1st , 2nd as well as 3rd trimester means of percentage chemotaxis. There significant decreases in the chemotaxis when compared to the non pregnant control(p <0.05).

Fig IV: Correlation graph of Total white blood cell count as well as percentage Viability shows that there was no significant negative correlation between Total Whiteblood cell count as well as percentage Viability ( p> 0.05)

Fig VII: Correlation graph of Total white blood cell count as well as percentage Nitroblue tetrazoliumShows negative correlation between Total white blood cell count as well as percentage nitrobluetetrazolium.( p >0.05)

Fig VIII: Correlation graph of Total white blood cell count as well as percentaghagocytosis . Shows that there are negative correlation between Total white blood cell count as well as percentage phagocytosis (p> 0.05).

Fig IX: Correlation graph of Total White blood cell count as well as percentage chemotaxis shows that there was negative correlation between Total whiteblood cell count as well as percentage chemotaxis ( p >0.05)

Fig X:Correlation graph of percentage chemotaxis as well as phagocytosis shows that there was significant positive correlation between percentage chemotaxis as well as phagocytosis(p < 0.05)

Fig XI : Correlation graph of percentage phagocytosis as well as Nitrobluetetrazolium shows that there was significant positive correlation between percentage phagocytosis as well as Nitrobluetetrazolium( p < 0.05)

Fig XII: Correlation graph of percentage nitroblue tetrazolium as well as percentage chemotaxis. shows that there was significant positive correlation between percentage nitrobluetetrazolium as well as percentage chemotaxis. ( p<0.05)

TABLE 1: THE MEAN AS WELL AS STAS WELL ASARD ERROR OF MEAN OF FULL BLOOD COUNT ANALYSIS IN CONTROL, PREGNANCY STATE, 1ST TRIMESTER, 2ND TRIMESTER AS WELL AS 3rd TRIMESTER PARAMETERS

s – Denotes significance (P <0.05) when controls are compared with pregnancy state

*Denotes significance (p <0.05) when controls are compared with 1st trimester

** Denotes significance (P <0.05) when controls are compared with 2nd trimester

***Denotes significance (P < 0.05) when controls are compared with 3rd trimester

PCV =Packed cell volume, HB = Hemoglobin, PLT= Platelet count, WBC= White blood cell count, N= Neutrophils, L= Lymphocytes, E-Eosinophils, M=Monocytes, B=basophils, VIA= Viability test, NBT=Nitrobluetetrezolium test, PHA= Phagocytic test, CHEM= Chemotaxis

TABLE 2: THE MEAN AS WELL AS STAS WELL ASARD ERROR OF MEAN OF LEUKOCYTE FUNCTIONAL ASSAYS IN CONTROL, PREGNANCY STATE, 1ST TRIMESTER, 2ND TRIMESTER AS WELL AS 3rd TRIMESTER PARAMETERS

Key

s – Denotes significance (P <0.05) when controls are compared with pregnancy state

*Denotes significance (p <0.05) when controls are compared with 1st trimester

** Denotes significance (P <0.05) when controls are compared with 2nd trimester

***Denotes significance (P < 0.05) when controls are compared with 3rd trimester

PCV =Packed cell volume, HB = Hemoglobin, PLT= Platelet count, WBC= White blood cell count, N= Neutrophils, L= Lymphocytes, E-Eosinophils, M=Monocytes, B=basophils, VIA= Viability test, NBT=Nitrobluetetrezolium test, PHA= Phagocytic test, CHEM= Chemotaxis

FIG I. BAR CHART COMPARINGCONTROL,PREGNANT STATE, IST, 2ND AS WELL AS 3RD TRIMESTER MEAN OF TOTAL WHITE BLOODCELL COUNT

FIG I; BAR CHART COMPARING CONTROL,PREGNANT STATE, IST, 2ND AS WELL AS 3RD TRIMESTER MEAN OF TOTAL WHITE BLOODCELL COUNT

There were significant increases( P < 0.05 respectively),in Total white blood cell count of pregnant women when compared with the non- pregnant controls

KEY

*Denotes significance at p< 0.05 when control was compared with pregnant state

**Denotes significance at p< 0.05 when 1st timesterwas compared with 2nd Trimester

*** Denotes significance at p< 0.05 when 1st timester was compared with 3rd

**** Denotes significance at p< 0.05 when 2nd timester was compared with 3rd Trimester

FIG II. BAR CHART COMPARING CONTROL,PREGNANT STATE, IST, 2ND AS WELL AS 3RD TRIMESTER MEAN OF PERCENTAGE VIABILITY

FIG II. BAR CHART COMPARING CONTROL,PREGNANT STATE, IST, 2ND AS WELL AS 3RD

TRIMESTER MEAN OF PERCENTAGE VIABILITY

There was no significant difference in the percentage viability of the pregnant as well as non pregnant controls(p > 0.05)

KEY

*Denotes significance at p< 0.05 when control was compared with pregnant state

**Denotes significance at p< 0.05 when 1sttrimester was compared with 2nd Trimester

*** Denotes significance at p< 0.05 when 1st timester was compared with 3rd

**** Denotes significance at p< 0.05 when 2ndtrimester was compared with 3rd Trimester

FIG III. BAR CHART COMPARING CONTROL,PREGNANT STATE, IST, 2ND AS WELL AS 3RD

. TRIMESTER MEAN OF PERCENTAGE NITROBLUETETRAZOLIUM

FIG III. BAR CHART COMPARING CONTROL,PREGNANT STATE, IST, 2ND AS WELL AS 3RD

. TRIMESTER MEAN OF PERCENTAGE NITROBLUETETRAZOLIUM

There were significantly decreased values when compared to the non pregnant controls(p< 0.05, respectively)

KEY

*Denotes significance at p< 0.05 when control was compared with pregnant state

**Denotes significance at p< 0.05 when 1sttrimester was compared with 2nd Trimester

*** Denotes significance at p< 0.05 when 1sttrimester was compared with 3rd

**** Denotes significance at p< 0.05 when 2nd timester was compared with 3rd Trimester

FIG IV. BAR CHART COMPARING CONTROL, PREGNANT STATE, 1st, 2ND AS WELL AS 3RDTRIMESTER MEANS OF PERCENTAGE PHAGOCYTOSIS

FIG IV. BAR CHART COMPARING CONTROL, PREGNANT STATE, 1st, 2ND AS WELL AS 3RD TRIMESTER MEANS OF PERCENTAGE PHAGOCYTOSIS

There weresignificant decreases( p < 0.05 respectively), in percentage phagocytosis when compared to the non- pregnant controls

KEY

*Denotes significance at p< 0.05 when control was compared with pregnant state

**Denotes significance at p< 0.05 when 1st timester was compared with 2nd Trimester

*** Denotes significance at p< 0.05 when 1st timester was compared with 3rdTrimester

**** Denotes significance at p< 0.05 when 2ndtrimester was compared with 3rd Trimester

FIG V. BAR CHART COMPARING CONTROL, PREGNANT STATE, 1st, 2ND AS WELL AS 3RD TRIMESTER MEANS OF PERCENTAGE CHEMOTAXIS

FIG IV. BAR CHART COMPARING CONTROL, PREGNANT STATE, 1st, 2ND AS WELL AS 3RD TRIMESTER MEANS OF PERCENTAGE PHAGOCYTOSIS

There were significant decreases in percentage chemotaxis(p <0.05, respectively)

KEY

*Denotes ignificance at p< 0.05 when control was compared with pregnant state

**Denotes significance at p< 0.05 when 1st timester was compared with 2nd Trimester

*** Denotes significance at p< 0.05 when 1st timester was compared with 3rd

**** Denotes significance at p< 0.05 when 2ndtrimester was compared with 3rd Trimester

FIG VI: CORRELATION GRAPH OF TOTAL WHITE CELL COUNT AS WELL AS

PERCENTAGE VIABILITY

FIG VI: CORRELATION GRAPH OF TOTAL WHITE BLOOD CELL COUNT AS WELL AS PERCENTAGE VIABILITY .

There was no significant negative correlation between total white blood cell count as well as percentage viability. ( P> 0.05)

FIG VII: CORRELATION GRAPH OF TOTAL WHITE CELL COUNT AS WELL AS

PERCENTAGE NITROBLUETETRAZOLIUM (NBT)

FIG VII: CORRELATION GRAPH OF TOTAL WHITE BLOOD CELL COUNT AS WELL AS PERCENTAGE NITROBLUETETRAZOLIUM . There was negative correlation between Total White blood cell count as well as percentage Nitrobluetetrazolium. ( P> 0.05)

FIG VIII: CORRELATION GRAPH OF TOTAL WHITE CELL COUNT AS WELL AS

PERCENTAGE PHAGOCYTOSIS (PHAG)

FIG VIII: CORRELATION GRAPH OF TOTAL WHITE BLOOD CELL COUNT AS WELL AS PERCENTAGE PHAGOCYTOSIS shows that there are negative correlation between Total White blood cell count as well as percentage phagocytosis. ( P> 0.05)

FIG IX :CORRELATION GRAPH OF TOTAL WHITE CELL COUNT AS WELL AS

PERCENTAGE CHEMOTAXIS (CHEM)

FIG IX: CORRELATION GRAPH OF TOTAL WHITE BLOOD CELL COUNT AS WELL AS PERCENTAGE CHEMOTAXIS. Shows negative correlation between Total White blood cell count as well as percentage chemotaxis. ( P> 0.05)

\

FIG X: CORRELATION GRAPH OF PERCENTAGE CHEMOTAXIS AS WELL AS

PERCENTAGE PHAGOCYTOSIS (PHAG)

FIG X: CORRELATION GRAPH OF PERCENTAGE CHEMOTAXIS AS WELL AS PERCENTAGE PHAGOCYTOSIS.There was significant positive correlation between percentage chemotaxis as well as percentage phagocytosis ( P < 0.05)

FIG XI: CORRELATION GRAPH OF PERCENTAGE PHAGOCYTOSIS (PHAG)

AS WELL AS PERCENTAGE NITROBLUETETRAZOLIUM (NBT)

FIG XI : CORRELATION GRAPH OF PERCENTAGE PHAGOCYTOSIS AS WELL AS PERCENTAGE NITROBLUETETRAZOLIYUM.

shows that there was significant positive correlation between percentage phagocytosis as well as percentage Nitrobluetetrazolium ( P < 0.05)

FIG XII: CORRELATION GRAPH OF PERCENTAGE NITROBLUETETRAZOLIUM

(NBT) AS WELL AS PERCENTAGE CHEMOTAXIS ( CHEM)

FIG XII: CORRELATION GRAPH OF PERCENTAGE NITROBLUETETRAZOLIUM AS WELL AS PERCENTAGE CHEMOTAXIS.shows that there was significant positive correlation between percentage nitrobluetetrazolium as well as percentage chemotaxis ( P < 0.05)

CHAPTER FIVE

DISCUSSION AND CONCLUSION

Pregnancy which is a state of natural transplantation has been shown to be immunologically dynamic particularly when the parameters in the various trimesters are compared.

The result of this study has shown a decrease in the Hemoglobin concentration as there was a difference in the values obtained from the non-pregnant women (P<0.05) with the women in their third trimester of gestation having the lowest values. This is in accordance with that observed by Beirnstein et al., (2001), that anaemia is relatively normal in pregnancy in which case is due to an increase in plasma volume in order to help supply oxygen as well as nutrients to both mother as well as baby thereby causing hemodilution.

There was significant elevation in total leukocyte count (p< 0.05) of the pregnant women in their various gestational ages when compared to the non-pregnant control. This is in synergy with the findings of chas well asra, et al., 2012 that leukocytosis occurs during pregnancy.This indicates that the total white blood cell count increases with gestational age as well as may be caused by physiological stress that was triggered by the pregnancy.

The differential white cell count, revealed a significantly increased neutrophil count( p < 0.05) when compared to the non pregnant women, as this is in line with the work of Gatti, 1994,that neutropils increase during pregnancy as well as often presents with toxic granulation as this may be due to impaired neutrophilic apoptosis in gestation. There was also a significant increase in monocyte count when compared to the values obtained in the non pregnant women , this is also, in line with the work of kline et al., 2005, that monocytes increase during pregnancy as this is to prevent fetal allograft rejection.However, Eosinophils as well as Basophils do not change significantly during pregnancy.

The Lymphocyte count was significantly decreased when compared to the non-pregnant women as this is in line with the findings of Kline, et al., 2005, that lymphocyte counts decreases during pregnany.This could be due to the increase in neutrophil production which is as a result of adequate bone marrow response to an increased drive for erythropoesis occuring during pregnancy.

Platelet count in this study, revealed a remarkable decrease in the values obtained in the pregnant women when compared with the non-pregnant control. This is in consonant with the findings of Shehlata, et al., 1999, that platelet count decreases during pregnancy. This is termed ‘Gestational thrombocytopenia” which could be partly due to hemodilution as well as also platelet activation as well as accelerated clearance.

There were significant decreases in the values obtained in the Leukocyte Functions (P<0.05, respectively).there was significant decrease in Nitroblue tetrazolium when compared to the non-pregnant control. This is in line with the work of Arinola, et al., 2004 that Nitrobluetetrazolium reduction is depressed during pregnancy .This could be due to reduced cellular immunity during pregnancy. Phagocytic ability of polymornuclear leukocyte represented by phagocytosis test was depressed in women during pregnancy when compared to the non-pregnant controls. . This result is in line with that of persellin as well as Leidfarth in 1978, who showed that neutrophil chemotaxis as well as phagocytic action are depressed, mostly due to inhibitory factors present in the serum of a pregnant female .There was significant decrease(p < 0.05) in chemotactic ability of Leukocytes during pregnancy when compared to the non-pregnant controls..This agrees with the findings of Krause,et al., 1987. This could be due to depressed cellular immunity which occurs especially during the late trimester of gestation.This indicates decreased Leukocyte functions with increasing ingestational age.

Correlation studies indicated that there was no significant negative correlation between the White blood cell count as well as the Leukocyte functions during pregnancy,(P >0.05,respectively indicating that increase in white blood cell count did not have any significant effect on leukocyte functions, therefore the increase may be as a result of physiologic stress induced by the gestational state. . However, there was significant positive correlation between chemotaxis, phagocytosis as well as Nitrobluetetrazolium (P< 0.05, respectively). This indicates that the ability for phagocytic leukocytes to carry out phagocytosis, is strongly affected by it's ability to undergo oxidative respiratory burst, as well as chemotaxis. Failing which, results in decreased leukocyte function. This could be due to immuno suppression so as to tolerate the foetus.

Immunosuppression which is a common occurrence in pregnancy is based on changing lymphocyte numbers as well as altered polymorphonuclear leukocyte functions. Which includes chemotaxis, engulfing as well as intracellular killing of invading pathogens (Bjorkson, 1978).

Early studies by Arinola, et al., 2004 have shown that immune status during early stage of pregnancy is dominated by Th 1 cytokines ( inducers of cell mediated immunity) but is replaced by Th2 cytokines ( inducers of humoral mediated immuniy) afterwards. Predominant humoral immunity expressed at the late stage of pregnancy explains depressed leukocyte migration during 2nd as well as 3rd trimesters.

The percentage chemotactic index , NBT reduction tests as well as Phagocytosis observed are similar to changes in parasitic infections as well as chronic granulomatous diseases, suggesting a common underlying mechanism.( El- mallam as well as Fletcher, 1980).

The findings of this study therefore, reveals that there is decreased Leukocyte functions with increase in gestational age suggestive of depressed cellular as well as humoral immunity resulting in susceptibility to infections. It is therefore imperative that adequate medical care as well as patient compliance is ensured during the period.

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APPENDIX

CONSENT FORM TO PARTICIPATE IN THE STUDY

STUDY NUMBER…….

PRINCIPAL INVESTIGATOR: ORIAZOWAN A. LIVETH

STUDY TITLE: LEUKOCYTE FUNCTIONS DURING PREGNANCY AMONGST NIGERIAN WOMEN IN EDO STATE.

INTRODUCTION

Leukocytes play a major role in host defence against foreign waders as well as infective agents, thereby building as well as maintaining the body’s immunity. Leukocyte chemotaxis (migration) is an essential factors for host defense. Therefore an impairment in carrying out this function may result in the patient becoming susceptible to infections.

PURPOSE OF STUDY

Oriazowan A. Liveth of University of Benin is the researcher in this study titled “Leucocytes functions in pregnant women attending ante-natal clinic in Stella Obasanjo Hospital, Benin City.

The aim of this in vitro research is to determine whether pregnancy exerts anti-oxidative, chemotactic, as well as phagocytic abilities of leukocytes in pregnant women as well as to what extend such effects contrast with that in normal subjects. The results obtain from the study may help better management during pregnancy. With this in mind, you are being requested to participate because you may be having leukocytes with defective functions. Therefore, it is good to know that if you chose to participate you will be better managed during conception as well as delivery

EXPLANATION OF PROCEDURES AS WELL AS TESTS

I have understood that when I agree to participate in this research, I will be faced with any of the following procedures based on my health status:

I will be asked a few personal questions related to my health.

I will have my agency to ask any relevant questions to the investigator.

I may undergo physical examinations

My blood sample may be taken from me

The samples will be permanently stripped of information that could identify me

My information will be kept confidential in accordance with State as well as Federal Law.

HAZARDS, RISKS AS WELL AS BENEFITS

I kno that asking questions as well as performing physical examinations will be safe and will cause no discomfort to me.

When withdrawing blood, one will feel slight pain at the puncture site but I have ensured that the pain will be slight as well as the risk of bleeding will be negligible.

In events when any complications is directly related to sample collection, the chief Medical officer will be available to aid assistance as well as informing the researcher.

I understand that personal benefit(s) to me major might not result from taking part in this study, but knowledge gained from my participation may benefit others.

I therefore participate at free will as well as not ask for payment.

CONSENT

Statement by participant: I have read as well as understood this research. Also, all my questions were answered satisfactorily. Therefore,

I consent to participate fully in this research.

I consent to participate voluntarily as well as may withdraw from the study any time I feel to do so.

I understand the information that was given to me concerning the research in my local language as well as have had all my questions answered to my satisfaction.

I also know that information I give will be treated confidentially.

A member of the team can undergo physical examination which will cause no harm to me.

Blood samples can be collected from me, and I know that I may feel slight pain at the site of the needle puncture.

During an unlikely event where complications may arise, the Medical officer will be available for assistance.

By signing this consent form, I agree to give sample to ORIAZOWAN A. LIVETH for research purposes.

_____________________________ _____________________________

Signature /Thumb of the participant Signature /Thumb of the Witness

______________________________

Signature of interviewer (Researcher)

ID No:______________ DATE:____________