i

DETERMINATION OF HAEMATOLOGICAL INDICES IN

PREGNANT AND POSTPARTUM WOMEN IN AYINKE HOUSE,

LAGOS STATE UNIVERSITY TEACHING HOSPITAL (LASUTH),

IKEJA.

A Dissertation submitted to the National Postgraduate Medical College of

Nigeria in partial fulfillment of the requirement for the award of the

Fellowship of the College in Obstetrics and Gynaecology.

By

DR OSHODI YUSUF ABISOWO

(MBBS IBADAN)

Department of Obstetrics and Gynaecology, Lagos State University

Teaching Hospital, Ikeja, Lagos.

ii

SUPERVISION

This work was carried out under the supervision of:

FIRST SUPERVISOR: DR OLUWAROTIMI I. AKINOLA MD, FWACS, FICS

Chief Consultant Obstetrician and Gynaecologist,

Department of Obstetrics and Gynaecology,

Lagos State University Teaching Hospital, Ikeja.

Signature: ………………………………………………

SECOND SUPERVISOR: DR ADETOKUNBO O. FABAMWO FWACS, FMCOG, FICS

Chief Consultant Obstetrician and Gynaecologist,

Department of Obstetrics and Gynaecology,

Lagos State University Teaching Hospital, Ikeja.

Signature: ………………………………………………

iii

CERTIFICATION

I hereby certify that this work was carried out by Dr. Oshodi Yusuf Abisowo of

the department of Obstetrics and Gyneacology, Lagos State University Teaching

Hospital, Ikeja, under our supervision.

DR. O. I Akinola

Department of Obstetrics and Gynaecology,

Lagos State University Teaching Hospital,

Ikeja, Lagos State.

DR. A.O FABAMWO

Department of Obstetrics and Gynaecology,

Lagos State University Teaching Hospital,

Ikeja, Lagos State.

Prof. A.A SOBANDE

Head of Department

Department of Obstetrics and Gynaecology,

Lagos State University Teaching Hospital,

Ikeja, Lagos State.

iv

DEDICATION

This book is dedicated to the Almighty God, the giver of l ife and

wisdom, for his guidance and support in all my endeavours. To my

parents and family for their support in my training and final ly to al l

the mothers that laid their l ives while performing the divine duty .

v

ACKNOWLEDGEMENT

I wish to express my profound gratitude to the Head of

Department and the entire Consultant staff of Obstetrics and

Gynaecology of the Lagos State University Teaching Hospital, for

their able supervision in carrying out this research.

I am particularly indebted to Dr O.I Akinola and Dr A.O

Fabamwo for their supervision of this work and fatherly advice. My

special appreciation goes to Dr O.Y Oyedele, Dr A. Tayo, Dr Mrs

Aina, Dr Mrs Ottun, Dr Gbadegesin, Dr Rabiu and Dr Adewunmi for

their guidance during my training. I acknowledge the contributions

of the entire residents of the department of Obstetrics and

Gynaecology, in the preparation of this dissertation.

vi

TABLE OF CONTENTS

Title and Author …………….…………………………......................... i

Supervisor …………………………………………………………………………. ii

Certification ………………………………………..………………………… iii

Dedication………………………………...…………….......................... iv

Acknowledgment ……………………………..……………………………… v

Table of contents …………………………………………………………….. vi

Summary ……………………………………………………………………….. vii

CHAPTER ONE

Introduction……………………………………..……………………………… 1

Aim and Objectives ………………………………… ……………………… 5

Rationale for the study ……………………………………………………. 6

CHAPTER TWO

Literature Review………………………………………………………………… 8

Red blood cell changes in pregnancy and puerperium ……….. 9

White blood cell changes in pregnancy and puerperium ……… 12

Platelet changes in pregnancy and puerperium ………………….. 13

Blood volume changes ……………………………………………………… 15

CHAPTER THREE

METHODOLOGY …………………………………………………………………… 19

Study site, Study duration and Study design …………………………. 19

Ethical Consideration ……………………………………………………………. 19

Recruitment of Patients …………………………………………………………. 20

Sample size Determination ……………………………………………………. 21

Sampling Technique ……………………………………………………………… 22

Inclusion criteria …………………………………………………………. 22

Exclusion criteria …………………………………………………………. 23

Clinical management …………………………………………………………….. 24

Principle of analysis ……………………………………………………………… 25

vii

Data Processing and Statistical Analysis ………………………………….. 26

Quality control ………………………………………………………………………. 27

Limitation of the study and Dissemination of information …………. 29

CHAPTER FOUR

Results ………………………………………………………………………………… 30

Tables and Figures ……………………………………………………………….. 38

CHAPTER FIVE

Discussion …………………………………………….................................. 48

Recommendations …………………………………………………………………. 57

References ……………………………………………………………………………. 58

CHAPTER VI

APPENDICES

Appendix I A and B – Participants informed Consent ………………… 72

Appendix II - Data collection form ……………………………….. 74

Appendix III - Laboratory form ………………………………………. 75

Ethics committee approval ……………………………………………………….. 76

viii

SUMMARY

Background:

Pregnancy is a time of considerable maternal adaptation during which the range

of normal laboratory values for commonly requested clinical investigations is

wider and even more arbitrary than in the non-pregnant state. The

haematological profile of an individual reflects the health status to a large extent

and indeed has been associated with pregnancy outcome. Although, pregnancy

specific values are becoming available due to increased awareness of the

obstetrician for their need, the available reference values are mainly based on

healthy Caucasians. Moreso, there is a dearth of information on the reference

values for haematological indices particularly in relation to the trimesters of

pregnancy and in the puerperium.

Objectives: To determine the reference values for various haematological indices

in trimesters of pregnancy and puerperium amongst booked healthy pregnant

and postnatal subjects in a large maternity unit (Ayinke House) in Lagos, South-

Western Nigeria.

Methods: This was a cross-sectional prospective study involving 422 subjects

carried out over a six-month period. Apparently healthy subjects that satisfied

the inclusion and exclusion criteria were recruited for the study and grouped into

six. There were 70 subjects in the first trimester group, 71 subjects each in the

second and third trimester groups and 70 subjects each in early puerperium, late

puerperium and non-pregnant groups respectively. Each subjects had her

anthropometry (weight and height) documented and haematological indices

determined by automated analysis using the Coulter counter method. The results

were subjected to statistical analysis using 95% confidence intervals.

ix

Results: The results from this study showed that the difference in the mean

haematocrit values in pregnancy was statistically significant between the first and

second trimesters (P=0.026) and also between the second and third trimesters

(P=0.007). The mean RBC count also showed a significant difference between

the first and second trimesters (P=0.032). Other red cell indices (MCV, MCH and

MCHC) showed no statistically significant difference in between trimesters. The

difference in the mean WBC count values was equally significant between the

first and second trimester (P=0.023). A similar significant difference was

observed in the mean platelet count between the first and third trimesters

(P=0.002).

Conclusion: Reference ranges of haematological indices have been described for

this environment. In this study, the lowest red cell parameters (haematocrit,

haemoglobin concentration and RBC count) were recorded in the second

trimester and there were varying statistically significant differences in the mean

haematocrit, haemoglobin concentration, RBC, WBC and platelet values during

the trimesters of pregnancy and the puerperium.

1

CHAPTER ONE

INTRODUCTION

Blood cell values are known to vary according to age, sex and race

amongst other factors1. Over the years, there are established differences

between the accepted normal adult haematological values derived from the

Caucasian population and values from healthy Nigerians2, 3, 4. In interpreting a

patient’s laboratory test results, the clinician usually compares the reported

values with the reference values. Data derived from unselected population or

population selected for disease state by virtue of having laboratory investigations

performed is likely to yield biased values. Even with the best of reference

standards, there will be an overlap of laboratory values of mildly affected

abnormal individuals into the normal range5, 6.

Pregnancy is a time of considerable maternal adaptation during which the

range of normal laboratory values for commonly requested clinical investigations

is wider and even more arbitrary than applied to non-pregnant state7. Successful

outcome of pregnancy requires large number of physiologic adaptations8. These

adaptations involve changes of metabolism in most organ systems resulting in

alterations of both haematological and biochemical composition of blood. In

order to be able to accurately interpret laboratory parameters of pregnant

women, physicians caring for them have to be aware of these physiological

changes that accompany pregnancy9.

2

Although there is a debate about the magnitude of change, there is

general agreement that red cell mass is increased in second and third trimesters

of pregnancy10, 11, 12. The control of red blood cell production is probably multi-

factorial but one important influence is the hormone erythropoietin. The

production of erythropoietin is dependent on tissue oxygen level in the kidneys

which in turn is governed by independent factors including atmospheric oxygen

tension, pulmonary function, cardiac output, red cell mass and the oxygen

affinity of haemoglobin13. During the first trimester of normal pregnancy, there

are changes in cardiac output and oxygen affinity in haemoglobin which would

tend to influence tissue oxygen levels in the kidney. Plasma erythropoietin level

is thus increased in pregnancy but the magnitude of the bone marrow response

to it, defined in terms of peripheral red blood cell count and mean cell volume

will depend on the level of iron supply to the marrow14, 15.

The haematological profile of an individual to a large extent reflects her

general health status16 and many studies have identified the haematological

profile of the pregnant woman as one of the factors affecting pregnancy and its

outcome17-22. The most common parameter referred to amongst the

haematological indices is an indicator of haemoglobin concentration and low

haemoglobin (anaemia) is the most widely identified hemoglobin abnormality23.

Maternal plasma volume increases by approximately 50% during first and

3

second trimesters of pregnancy whereas the corresponding increase in red cell

mass is only 20-30% giving rise to a state of physiological anaemia more

profound in mid-pregnancy 13, 24-26.

Although pregnancy specific values are becoming available due to

increased awareness of the obstetricians for their need13, the available reference

values have been mainly those based on healthy Caucasians27. Reference values

for pregnant women have been proposed based on few studies in Nigeria and

the tropics28, 29. Conflicting results have been documented regarding the

statistical significance of variations in some of these haematological parameters

at different trimesters. While some workers did not find any significant change

between the second and third trimesters with respect to any of the

haematological parameters studied amongst Nigerians29, 30, Onwukeme and

Uguru28 reported differences in total white cells and neutrophils counts in similar

subjects. This difference was attributed to environmental factors, ethnic and

tribal peculiarities31. The role of parity came into fore when Onwukeme in 1992

noted that variations in haemoglobin, heamatocrit, leucocytes and platelet counts

in the puerperium were more marked in primipara than multipara32.

Racial component to this variation of haematological values has been

observed in various parts of the world. High proportion of low haemoglobin

concentration was found in Black and Indian populations compared to Whites

4

and Orientals living in the United Kingdom33. Analysis of data from two major

nutritional surveys showed a mean haemoglobin level in Blacks 1g/dL less than

Whites34, 35. This difference was independent of age, economic level and

nutritional status36, 37. Essien et al4 also found lower platelet count in adult

Nigerians than in Caucasian living in Nigeria. Flemin and Harrison found that total

white cell and neutrophils counts were lower in Nigerians than the Caucasians38.

The available reference values based on healthy Caucasians may therefore, not

be appropriate for our own population due to well recognized nutritional,

environmental and genetic factors39. Recent changes in nutritional status, dietary

habits, environmental factors and economy of the country may influence the

haematological indices of present day pregnant Nigerians.

In the light of the above, it is desirable to investigate the reference values

for our pregnant and puerperal population to assess the conformity of same with

those established references in the literatures. Such a study is of importance

since antenatal care and pregnancy outcome is in part predicated on monitoring

of and response to these haematological indices.

This study is therefore designed to present the range of variation in

haematological values in apparently healthy pregnant women attending a large

maternity hospital in Lagos, Southwestern Nigeria.

5

AIM AND OBJECTIVES OF THE STUDY

Main Aim

To determine the reference values for various haematological indices in

pregnancy and puerperium amongst booked healthy ante-natal and post natal

subjects in Department of Obstetrics and Gynaecology (Ayinke House), Lagos

State University Teaching Hospital, (LASUTH), Ikeja, Lagos.

Specific Objectives

1. To determine haematological profile in pregnant women in the three

trimesters of pregnancy.

2. To determine baseline haematological indices in early and late puerperium

3. To compare values obtained from pregnant and puerperal subjects with

age-matched non-pregnant women

4. To determine whether data obtained 6 weeks postpartum were

representative of the non-pregnant state.

6

RATIONALE FOR THE STUDY

Nigeria has an extremely high maternal mortality rate (MMR)40 with a rate

of 704 per 100, 000 live births, implying that with about 2.4 million live births

annually, some 170,000 Nigerians die as a result of complications associated with

pregnancy or childbirth. The most common direct obstetrics causes of death in

Nigeria include haemorrhage, sepsis, pre-eclampsia/eclampsia and anaemia41, all

of which manifest alteration of normal haematological indices. Therefore, women

who are at higher risk of morbidity and mortality need to be identified early so

that appropriate and timely measures can be taken.

The maintenance of normal blood counts during gestation is very relevant

in the overall wellbeing of the woman and the reproductive health status of a

nation. It is also a very useful basic tool in prediction of morbidity and mortality

in women of childbearing age group. However, there is paucity of information in

this field in Nigeria and the reference values have been mainly based on healthy

Caucasians. Defining normal pregnancy ranges will help in interpreting test

results in pregnancy since comparison between normal values derived from non

pregnant subjects will lead to bias.

It is therefore the intention of this study to evaluate and describe the

haematological profile of healthy pregnant and postpartum women in our

environment to obtain relative baseline values and compare these with the

established ranges in the literature for conformity. It also affords the

establishment of differences between pregnant and age-matched non-pregnant

7

controls, possible variations among the three trimesters and in early and late

puerperium. It also allows the defining of normal ranges for healthy subject that

can be used as reference in infective or other diseased states.

8

CHAPTER TWO LITERATURE REVIEW

The cellular elements of the blood (white blood cells, red blood cells and

platelets) are suspended in the plasma. In the adult, these cells are formed in

the bone marrow whereas they are produced in the liver and bone marrow of the

fetus42. Normally 75 percent of the cells in the marrow belong to the white blood

cell producing myeloid series while only 25 percent are maturing the red cells,

even though there are over 500 times as many red cells in the circulation as

there are white cells. This difference in the marrow reflects the fact that the

average life span of the white cells is short (a few days to one week) whereas

that of the red cells is long43 (120 days).

RED BLOOD CELLS INCICES

Red blood cells are erythrocytes that carry haemoglobin responsible for

tissue oxygenation in the circulation. The red cell count in the adult non-

pregnant female is about 4.8 million per microlitre with life span of about 120

days. Each red blood cell contains approximately 29 pg of haemoglobin. The red

cell indices include heamatocrit (Hcrt), haemoglobin concentration (Hb), mean

corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean

corpuscular haemoglobin concentration (MCHC) 42. These indices are indicators

of haemoglobin concentration and low haemoglobin described as anaemia is the

most widely identified haemoglobin abnormality23

9

Changes in haematological indices in pregnancy may be due to several

factors including placenta hormones44, increased erythropoietin production and

increase in plasma volume 45. The increasing plasma volume however produces

apparent reduction in the haemoglobin concentration which is a normal

physiological response to pregnancy46.

CHANGES IN RED BLOOD CELL INDICES

Haematocrit, haemoglobin concentration and red cell count

It was observed that by 12 weeks gestation that the haematocrit and

haemoglobin concentration had decreased significantly below the non-pregnant

level13. This decrease was explained by the dilutional effect of increased plasma

volume alone unrelated to any change in the red cell mass. Haematocrit and

haemoglobin concentration progressively fell till 26-30 weeks gestation and

thereafter rise progressively to term attaining early pregnancy values13. This

finding was consistent with that of Akingbola et al39.

In other studies of Nigerian population using manual method of analysis,

Obisesan et al29 found no significant change in the red cell indices during

pregnancy while Dapper et al31 in Port-Harcourt found significant reduction in the

haematocrit only. It will be seen from this that no single value can be taken as

normal throughout pregnancy and this should be noted when diagnosing

anaemia. Though anaemia is defined by the World Health Organization as

Haemoglobin concentration less than 110g/L47, any haemoglobin reading less

10

than 105g/L at any point in pregnancy should be considered anaemia and

warrant further investigations and treatment48.

The red cell count also fell during pregnancy in mid-trimester relative to

early pregnancy level. Subsequently, it rose with gestation after 30 weeks from

till term13. The rise of haematocrit, haemoglobin concentration and red cell count

in third trimester may be due to reduced plasma volume expansion which is

more pronounced in iron supplemented pregnant women. It was also noted that

pregnant women taking iron supplementation have higher haematocrit,

haemoglobin concentration and red cell count than non-supplemented

women49,50. This implies that beside haemodilution, iron deficiency erythropoiesis

is a contributor to the lower haemoglobin values in pregnant women not using

iron supplement51. While poor nutritional status may contribute to low

haematocrit in pregnant Nigerian women52, iron therapy has been found to

improve the heamatocrit levels in pregnant Nigerian women53.

Crawford (1940) and Dennis (1976) reported that the heamatocrit,

already low during normal pregnancy decreases further during first 4 days post-

delivery, thereafter progressively increasing to non-pregnant values54,55. This

puerperal decrease may reflect the changes in the plasma volume during this

time. The already expanded volume which reaches maximum during the third

trimester of pregnancy is further increased by 1200ml during the first 4 days

following delivery43, 56. De Leeve et al did not find an increase in the red blood

11

cell in their puerperal subjects though the individual with largest increase in the

red cell mass during pregnancy has shorter red cell survival times57.

Hence it could be that selective destruction of the larger red cells allied to

a transient increase in plasma volume during early puerperium accounts for the

initial fall in haemacrit. While Taylor and Lind7 observed that the effect of

pregnancy on these red cell indices were persistent because values obtained at 6

weeks post-partum were lower relative to those obtained at months after

delivery, Onwukeme32 reported similar values for 6 weeks post-partum and non-

pregnant subjects.

Changes in mean corpuscular volume (MCV), mean corpuscular

haemoglobin (MCH) and mean corpuscular haemoglobin concentration

(MCHC)

The MCV and MCH had lowest values in first trimester, rose to the highest

level in second trimester and then declined again in third trimester. Hamilton and

Davidson also noted a gradual rise in MCV and MCH from early pregnancy to

term58. The MCV in their study rose significantly from 83.62fL in first trimester to

89.06fL at term whereas that of MCH was not significant. However, similar rise

were noted in these two indices by Milman et al 59 from 18th to 32nd week of

pregnancy but thereafter remained constant till term before falling again in the

puerperium.

12

It was suggested that MCV may be a better index of anaemia during

pregnancy than haematocrit and haemoglobin concentration both of which are

reduced by increased plasma volume. There was slight but insignificant change

in the MCHC throughout pregnancy60. While, the MCV gradually reduces to non-

pregnant level at the end of puerperium, there was no significant change in

MCH/MCHC.

WHITE BLOOD CELL CHANGES

The white blood cells are important in immune regulation and wound

healing. The count in peripheral circulation is about 4-11,000 per microlitre in

Caucasians27 but lower values have been reported for African population2, 3.

During pregnancy, there is marked increase in white cell count from an average

of 7,000 per microlitre in non-pregnant state to 10,500 per microlitre in late

pregnancy48.

Leucocytosis due to neutrophilia during normal pregnancy was recognized

as early as 19th century61. Leucocytosis due to neutrophilia is also well recognized

in Nigerian women irrespective of their genotype52.However, its pattern and limits

are not uniform61-63. Leucocytes and neutrophils gradually and significantly

increase from first to third trimester59,64. However, Taylor and Lind ‘8265 noted a

rise in leucocyte count until 34 weeks gestation followed by a small decrease

during the last month of pregnancy.

13

Various factors associated with leucocytosis in pregnancy include elevated

oestrogen secretion, raised plasma cortisol and increasing parity61-63. Awodu et al

speculated that subclincal infection may be responsible for significant fall in

heamatocrit and leucocytosis in pregnancy52.

Lymphocytes do not show any significant variation in pregnancy

according to findings of Griffin66 and Onwukeme28 but Awodu et al observed that

lymphocyte count decreased in second trimester52. Monocytes count exhibit

tendency to rise during the last third of pregnancy62,64 whereas both eosinophil

and basophil decline significantly during pregnancy.

Griffin and Beck in 1980 found significant increase in total number of

Leucocytes and neutrophils at delivery and for the first 24 hours postpartum66.

This rise might be attributable to normal acute inflammatory response to

placenta delivery and episiotomy site in early puerperium. Since an increased

white cell count is universal in early puerperium, it may not be justifiable to

interpret leucocytosis alone at this time as being indicative of infection7 without

considering the clinical features. The lymphocyte count rose slightly but

insignificantly a few hours after delivery and subsequently returned to normal.

PLATELET CHANGES

The platelets are non-nucleated blood cells. They are small granular

bodies that aggregate at the site of vascular injury. The peak platelet count lasts

14

about two weeks because the lifespan of platelet is about 8 to 11 days67. While

some reports suggested that platelet count decreases as the pregnancy

advances68-70, others reported stable platelet count throughout pregnancy71,72.

However, Mor et al69 found progressive increase in platelet count during

pregnancy among their subjects.

Hence, individual patient’s value varies: some increase, others decreasing

and some not showing any change at all, so no generalization about change in

platelet count during pregnancy can be made73. The fall in platelet count during

pregnancy may be explained by the dilutional effect of relative increase in

plasma volume assuming platelet production is fairly constant during

pregnancy74. This probably reflects an increased platelet turnover which would

support the idea that platelet survival time is decreased during normal

pregnancy67,75.

The platelet count increases significantly during the postpartum period76

and reaches about 60 percent above the late gestational value77-79 by the end of

the first week. This temporary increase in platelet count after delivery must be

considered as a compensatory increase in platelet production after a period of

platelet consumption during separation and delivery of the placenta8. The

platelet count thereafter rises and approaches the non-pregnant level by 6 weeks

postpartum32. The higher platelet count may contribute to increased risk of

thrombo-embolic complications in the puerperium.

15

The spleen is an important filter that removes spherocytes and other

abnormal red cells. It also contains many platelets and plays a significant role in

the immune system. Abnormal red cells are removed when they lose their

elasticity which prevent them from squeezing through the slits between

endothelial cells that line the splenic sinusoid80.

BLOOD VOLUME CHANGES

Physiological changes during pregnancy facilitate the adaptation of the

cardiovascular system to the increased metabolic needs of the mother, thus

enabling adequate delivery of oxygenated blood to peripheral tissues and the

fetus. Women without heart disease adapt well and adverse cardiac events are

rare. In some women, heart disease may first be detected during pregnancy

when inadequate adaptation exposes previously unrecognized limitations of

cardiac reserve. In the presence of important maternal structural heart disease,

increased cardiovascular demands of pregnancy can result in cardiac

decompensation, arrhythmias, and, rarely, maternal death.

Changes in the antepartum period

An increase in blood volume and heart rate as well as a reduction in

systemic vascular resistance bring about the increase in cardiac output necessary

to sustain pregnancy. An increase in blood volume has been documented in a

16

number of studies; however, there is variability among studies with regard to the

magnitude and timing of this increase.

Blood volume begins to increase in week 6 of gestation and by the end of

pregnancy it will have reached approximately 50% more than in the pre-

pregnant state24,81. Individuals differ considerably; one study demonstrated

individual increases from 20% to 100% above pre-pregnant blood volume82. All

studies have shown that blood volume progressively increases, at least until mid-

pregnancy; some studies have found that it plateaus in the third trimester83,84,

whereas others suggest that it increases continuously until term85. The increase

in blood volume is more pronounced in twin pregnancies86. Red cell mass

increases as much as 40% above pre-pregnancy levels24,84.

The plasma volume increase is proportionally greater than the increase in

red blood cell mass, and the resulting haemodilution explains the so-called

‘physiological anemia of pregnancy’. Reduced plasma volume expansion has

been associated with low birth weight and intrauterine growth retardation87.

Changes in the peripartum period

Pain, anxiety and uterine contractions all alter the haemodynamics at the

time of labour and delivery. Increases in cardiac output, tachycardia and

hypertension may stress the marginally compensated woman with heart disease

and also lead to decompensation. During the first stage of labour uterine

contractions can increase central blood volume by as much as 500 mL, the so-

17

called ‘auto-transfusion88. On average, vaginal delivery results in the loss of 10%

and cesarean delivery in 29% of total blood volume, although there is wide

individual variation in the magnitude of blood loss89. Placental separation does

not usually cause any significant change to the circulation. The basal blood

pressure increases during labour and further increases with each uterine

contraction; this is thought to result partly from the increasing cardiac output. In

addition, compression of the lower limb vessels may redistribute blood to the

upper limbs and add to upper body hypertension.

Changes in the postpartum period

A few hours after delivery, two-third of the pregnancy hypervolaemia

remained. This reduction could be accounted for almost completely by blood at

or soon after delivery. One week later, the pregnancy induced hypervolaemia

had been further reduced by another one third. This results in haemo-

concentration. During the next several weeks, the blood volume returns to

normal non-pregnant level with further haemo-concentration

Haemodynamic parameters slowly return to baseline values, but full

resolution may take as long as 6 months after delivery. Thus, haemodynamic

studies that used early postpartum values as surrogates for pre-pregnancy

baseline values, when the latter were unavailable, may underestimate the actual

change that occurs during pregnancy90.

18

While acknowledging local studies relevant to the topic of this research,

some limitations were observed which this study intends to improve upon.

Most of the studies were based on manual method of laboratory analysis of

haematological parameters except Akingbola et al39. This previous techniques

might have estimated haematocrit incorrectly since centrifuged red cell column

always contain between 2-6% of trapped plasma39. Other inaccuracies include

inability to obtain a flat seal at the bottom of the haematocrit tube and relative

difficulty in reading the height and column of the red cells.

The sample size when adequate is not uniformly distributed across the

trimesters of pregnancy29,31,39 while a few others have scanty or no first trimester

subjects at all29,39. In the 314 pregnant subjects in Akingbola’s study, only 9

subjects were recruited in first trimester compared to 124 and 181 in second and

third trimesters respectively.

The exclusion criteria sometimes were not fully explained as some

variables as grandmultiparity, blood transfusion, multiple pregnancy in index

pregnancies were possibly not considered.

Considering the above findings in local literature, the present study was

formulated with similar sample size across the trimesters of pregnancy and

puerperium. It employs strict exclusion criteria to remove confounding variables.

Detailed analysis of complete blood count using automated Coulter Counter

method to obtain accurate haematological indices which will serve as reference

range of values for the pregnant and postpartum women in this environment.

19

CHAPTER THREE

METHODOLOGY

STUDY SITE

The study was carried out among women attending ante-natal and post-

natal clinics and those parturients in lying-in ward of the Department of

Obstetrics and Gynaecology, Lagos State University Teaching Hospital, Ikeja,

Lagos in South-western part of Nigeria. About 15 new cases were booked every

day of the week. The delivery rates were average of 340 births monthly and

4000 births per year.

STUDY DURATION

Patients were recruited consecutively until the desired sample size is

attained. The study duration lasted six months between 3rd of January 2010 to

30th of June 2010.

Study Design

The study was prospective, cross-sectional study involving healthy

pregnant and post-partum subjects. These were compared with age-matched

non-pregnant subjects used as control.

Ethical Considerations

Approval for the study was obtained from the Ethics Committee of Lagos

State University Teaching Hospital, Ikeja, Lagos State (Appendix IV). The

20

subjects for the study were fully briefed on the research protocol in the language

they understand (Appendix IA and IB).

They were informed that information gathered will contribute to the

knowledge of haematological indices in our environment which will serve as

normal reference values in healthy state. They were encouraged to ask question

on any aspect of the study. They were also informed of the right of refusal to

take part in the study or withdraw at any point during the study which will not

affect the standard of their care in anyway. They were assured that all

information given will be treated as confidential. Benefit of the research was

also highlighted as it will allow patient to know their current haematological

indices. Those whose results were found to be below normal were referred back

to the clinic for proper investigation and appropriate management to correct

same. Informed written consent was then obtained (Appendix I A and B).

Recruitment of patients

Consecutive pregnant subjects that were attending the ante-natal clinic,

parturients in lying-in ward and those attending the post-natal clinic for follow-up

and have satisfied the inclusion criteria were recruited for the study. All pregnant

women were routinely placed on iron supplementation (ferrous sulphate 200mg

thrice daily) and folic acid 5mg daily from the second trimester. Malaria

chemoprophylaxis with sulphadoxine-pyrimethamine combination was given after

quickening and six weeks later according to our institutional policy.

21

The gestational age was derived form the last normal menstrual period

(LMP) or early ultrasound scan dating of the pregnancy where the LMP is not

known. Healthy non-pregnant controls were recruited from those patients

presenting at family planning clinic for the first time that met the inclusion

criteria. Information on each study subject was recorded in a designed proforma

attached (Appendix II).

Sample Size Determination

Sample size for single proportion

n = Z2.p. (1-p)

d2

n = sample size required for the study

Z = the standard normal deviate, usually 1.96 at 95% confidence level

p = prevalence rate (50% was used in this case)

d = precision rate (5%); degree of accuracy required

Type of test = two-sided test

n = (1.96)2 x 0.5 x (1 – 0.5)

(0.05)2

= 383

A sample size of 383 was required for the study.

In order to accommodate possible attrition or unforeseen errors in

completing the study questionnaire or blood sample processing, an additional

22

10% (39 subjects) of the calculated figure were recruited to bring the figure to

422 subjects.

SAMPLING TECHNIQUE

Stratified sampling (a probability sampling technique) was used to recruit

subjects, following our inclusion and exclusion criteria. Six groups were observed

in all; First trimester (up to 14 weeks gestation), second trimester (15– 28

weeks), third trimester (29weeks till term), early puerperium (2 – 4 days

postpartum), late puerperium (6 weeks postpartum) and non-pregnant subjects

(control).

Convenience sampling method (a non-probability sampling technique) was

be used to recruit subjects within the different group as patients were recruited

consecutively until the desired sample size was attained. Seventy-one subjects

were recruited in the second and third trimester groups while 70 subjects were

recruited each for first trimester, early puerperium, late puerperium and non-

pregnant subjects making up the total 422 subjects as determined by the sample

size calculated.

Sampling procedure

Inclusion criteria

a) Pregnant Subjects: All apparently healthy women with no adverse

medical/obstetrics history that satisfy the inclusion criteria were recruited

into the study.

23

b) Post-natal subjects: those that have vaginal delivery with estimated blood

loss less than 500ml.

c) Non-pregnant Subjects: healthy women within the reproductive age

group.

Exclusion criteria

a) Denial of consent

b) Febrile illness in the last four weeks

c) Features or laboratory diagnosis of haemoglobinopathy or bleeding

disorder

d) Chronic medical ailment

e) Bleeding in pregnancy (threatened abortion or ante-partum haemorrhage).

f) Hypertensive disease in pregnancy including pre-eclampsia and eclampsia

g) Diabetes mellitus

h) Blood transfusion

i) Multipara with last childbirth or miscarriage less than two years

j) Multiple pregnancy

k) Grandmultipara

l) Patients who were delivered by caesarian section

m) Acute renal, liver or systemic diseases acquired during pregnancy

24

Clinical management

With the patient sitting in upright position, a tourniquet was applied for a

few seconds (with minimal stasis) and 3ml of venous blood is drawn from the

antecubital vein by means of venepunture into the vacutainer tubes containing

di-potassium ethylene di-amine tetra-acetic acid (K2-EDTA). Being a cross-

sectional study, blood samples were collected from different subjects at 8-14

weeks in first trimester, 22-28 weeks in second trimester and 34-40 weeks in

third trimester. Early post partum samples were taken 2-4 days post delivery in

lying-in ward. Late postpartum samples were obtained in the postnatal clinic six

weeks after delivery. Healthy non-pregnant subjects were recruited among first

attendees at the family planning clinic that met the inclusion and exclusion

criteria.

Samples were collected between 1100 and 1300 hours and refridgerated

after proper labeling for identification. They were analyzed within 2-4 hours of

collection. Sample analysis was done at the Research laboratory of the

Department of Obstetrics and Gynaecology at Lagos State University Teaching

Hospital using BC-3000 Haematology Analyzer Model 2003-2005( Shenzhen

Mindray Bio-medical Electronic Company Limited. China).

Indices of measurement included the haemoglobin concentration (Hb),

heamatocrit (Hcrt), mean corpuscular volume (MCV), mean corpuscular

haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC),

white blood cell count (WBC) with differentials neutrophil and lymphocyte count,

25

red blood cell count (RBC) and platelet count (Platelet), all of which were

determined by auto-analysis. Results were recorded in the laboratory form

(Appendix III).

PRINCIPLE OF ANALYSIS

Mindray Auto-Haematology Analyser was used to provide an automated

complete blood count by Coulter Counter method.

Principle: A suspension of blood cells was passed through a small orifice

simultaneously with an electric current. The individual blood cells passing

through this orifice introduces an impedance change in the orifice determined by

the size of the cells and provides cell size distribution. The red blood cells and

the platelets were then counted. The haematocrit was also measured.

The lytic reagent (Lyse) used for the complete blood count parameters

prepares the blood so the system can count and measure the amount of

haemoglobin. The lytic reagent rapidly and simultaneously destroys the

erythrocytes and converts the haemoglobin to a stable cyanide-containing

pigment while it leaves the leucocytes nuclei intact. This pigment is measurable

at 525nm, the absorbance of which is directly proportional to the haemoglobin

concentration of the sample. Other red cell indices (MCV, MCH, MCHC) were

derived from the haematocrit and haemoglobin concentration.

The reagents used were Diluents, Lytic reagent and Cleaning agent.

Diluent is an isotonic electrolyte solution that dilutes the whole blood

samples, stabilizes cell membrane for accurate counting and sizing, conducts

26

aperture current, rinses instrument components between analysis, carries and

focuses the sample stream in the flow cell to direct the blood cells through the

aperture.

Lytic reagent rapidly lyses erythrocytes, free haemoglobin and reducing

the cellular debris to a level that does not interfere with leukocyte count.

The Cleaning agent cleanses and rinses the internal surfaces of the

instrument components after each cycle of analysis. These components are the

sample probe, the WBC bath, RBC and platelet bath and metering tube.

Data processing and statistical analysis

Data were analyzed using SPSS version 16.0 (Statistical Package for Social

Sciences, Inc., Chicago, Illinois); a statistical computer software. Descriptive

statistics (minimum, maximum, mean, and standard deviation) were determined

for all haematological indices and other appropriate variables. Proportions and

percentages were calculated for categorical variables.

One-way analysis of variance (ANOVA), a parametric inferential statistical

procedure was used to compare the means of haematological indices in the

different groups of patients (Non pregnant, 1st trimester, 2nd trimester, 3rd

trimester, early puerperium, and late puerperium).

Bonferroni Post Hoc Multiple Comparison was used to compare difference

within variables during pregnancy and puerperium with reference to non-

27

pregnant subjects. P-value less than 0.05 was considered to be statistically

significant at 95% confidence level.

Quality control of Auto-Haematology Analyser

In order to measure the reliability of the auto analyser, Cronbach’s alpha,

a measure of reliability was used. Alpha coefficient, an indicator of internal

consistency and accuracy of data sets was determined. Observations were taken

3 times for each of the haematological indices. A total of 20 patients were used

for this control analysis.

Haematological

Indices

Cronbach’s Alpha Co-

efficient

PCV 0.924

HB concentration 0.867

MCV 0.845

MCH 0.911

MCHC 0.898

RBC 0.941

WBC 0.956

Neutrophil 0.899

Lymphocyte 0.885

Platelets. 0.835

28

Testing the internal consistency of the observations within each parameter,

Cronbach’s alpha showed a high level of consistency (accuracy) in the

haematological indices, all ranging from 0.835 to 0.956. The closer the alpha co-

efficient to 1, the more reliable the value of the parameter measured.

29

LIMITATIONS OF THE STUDY

This study has a few limitations. Firstly, it was a cross-sectional study. A

longitudinal study may be more desirable because observed changes and trends

during pregnancy and puerperium might have been more accurate. It would

have also excluded unobserved individual differences in the study population.

Secondly, the sample studied is hospital-based and findings obtained from

this study can only be applicable to institutional-based patients. The findings in

this study may not be applicable to the general population.

Despite these limitations, the study probably provides an accurate picture

of the range of haematological values expected of apparently healthy pregnant

and postpartum women attending Ayinke House Maternity Hospital in Lagos.

DISSEMINATION OF INFORMATION

The patients were given scheduled appointment to see the researcher for

their results which were written out separately by the laboratory technologist.

Those with normal results were reassured and encouraged to continue their

current care. Those with abnormal results for example; anaemia were referred

back to the managing unit in Obstetrics and Gynaecology department where

thorough evaluation like blood film for malaria parasite, peripheral blood film

comment, stool for ova and cyst of parasites and occult blood, stool and urine

microscopy for culture and sensitivity were carried out. They were subsequently

managed as appropriate based on the result of the investigations.

30

CHAPTER FOUR

RESULTS

During the study period (3rd of January 2010 to 30th of June 2010), a

total of 422 subjects who had complete blood count examination and

satisfactorily filled the data collection form were recruited for the study. The

subjects were grouped into six: 70 in first trimester, 71 each in second and third

trimesters and 70 each in early and late puerperium and 70 non-pregnant

subjects who served as control.

The age of the subjects studied ranges from 17 to 41 years while the

mean values and the standard deviations and ranges for age, weight, height and

body mass index of all subjects were as shown in Table I. There was statistically

significant difference in only the weight across all groups (P=0.032) in Table I.

The subjects in the third trimester group had the highest mean weight

(75.34+13.80kg) while the non-pregnant subjects had the least mean weight

(62.70+11.60kg). Age, height and body mass index did not show significant

difference across the six groups (Table I).

One hundred and twenty-three subjects were para 0, 153 subjects were

para 1, and 84 subjects were para 2. The remaining 72 subjects were para 3 and

4. Ninety-six percent of the subjects were married, 74.4% had tertiary education

while 20% had secondary education. 70.4% of the respondents were from the

Yoruba tribe, 16.4% were Igbo and 9.8% were from the Minority group. Only

31

3.6% respondents were Hausas. 76.8% of the subjects were Christians and

23.2% were Muslims.

HAEMATOCRIT (Packed Cell Volume)

The haematocrit decreased gradually from 31.98 + 2.31% in first

trimester to 30.24 + 3.68% in the second trimesters but increased to (32.18 +

3.19%) in third trimester (Table II). It dropped further in early puerperium to

(31.49 + 3.71%) but gradually rose to (36.10 + 3.25%) obtained in the late

puerperium (Table III). The corresponding value for non-pregnant subjects was

33.84 + 3.39% (Figure I).

A total of 233(55.2%) subjects had anaemia (51% mild and 4%

moderate) using the haematocrit of 33% as benchmark. The breakdown of

anaemic subjects in different groups revealed 55% of those in first trimester,

61% among second trimester and 52% in third trimester subjects. The

remaining were 53% of those in early puerperium, 24% of those in late

puerperium and 42% of the non-pregnant subjects. None of the subject had

severe anaemia. Comparison of the mean haematocrit values for all groups

using analysis of variance (ANOVA) was statistically significant with P= 0.001.

HAEMOGLOBIN CONCENTRATION

The mean haemoglobin concentration decreased gradually from 10.22 +

0.88g/dl in first trimester to 9.63 + 1.25g/dl the second trimesters but

32

subsequently increased to 10.18 + 1.14g/dl in third trimester (Table II). It

declined in early puerperium marginally to 10.13 + 1.28g/dl and thereafter

increased progressively to 11.37 + 1.27g/dl in the late puerperium (Figure II).

The mean haemoglobin concentration was 10.50 + 1.50 g/dl in non-pregnant

subjects.

Following the analysis of haemoglobin concentration, 66 (15.6%) subjects

had moderate anaemia and 222(52.6%) had mild anaemia using the WHO

criteria. Comparison of the mean haemoglobin concentration values for all

groups (ANOVA) was statistically significant with P= 0.001.

MEAN CORPUSCULAR VOLUME (MCV)

The mean corpuscular volume changed little in the subjects studied. The

MCV in the first trimester (81.13 + 4.84fl) increased to 83.67 + 5.54fl and 84.41

+ 7.10fl in second and third trimesters respectively (Table II). After delivery, the

MCV dropped to (82.86 + 7.12fl) in early puerperium and (82.68 + 5.94fl) in late

puerperium which was close to the non-pregnant value of 82.41 + 5.57fl (Figure

III). With the P value of 0.222, the mean values of the MCV in all groups showed

no statistically significant difference.

MEAN CORPUSCULAR HAEMOGLOBIN (MCH)

The mean corpuscular haemoglobin increased slightly from 26.26 +

2.00pg in the first trimester to 26.55 2.70+ pg and 26.60 + 2.91pg in second

33

trimester and third trimesters respectively (Table II). After delivery, it gradually

declines to 26.30 + 3.13pg in early puerperium and 26.06 + 2.53pg in late

puerperium which was higher than 25.52 + 2.78pg in non-pregnant subjects.

(Figure IV). Comparing the trend in the mean corpuscular haemoglobin in all

groups, it was not significant at the P value of 0.189.

MEAN CORPUSCULAR HAEMOGLOBIN CONCENTRATION (MCHC)

The MCHC decreased from 32.01 + 1.54g/dl in the first trimester to 31.73

+ 1.72g/dl and 31.59 + 1.45g/dl in the second and third trimesters respectively

(Table II). The MCHC thereafter increased slightly to 31.85 + 1.83g/dl in early

puerperium but dropped again to 31.57 + 1.68g/dl in late puerperium which was

higher than 31.07 + 2.04g/dl in non-pregnant subjects (Figure V). Comparing

the trend of the means of corpuscular haemoglobin concentration in all groups, it

was statistically significant with the P value of = 0.033.

RED BLOOD CELL COUNT (RBC)

The red blood cell count decreased from 3.88 + 0.28 × 106/μl in the first

trimester to 3.64 + 0.49 × 106/μl in second trimesters and increased marginally

thereafter to 3.84 + 0.51 × 106/μl and 3.86 + 0.53 × 106/μl in third trimester

(Table II) and early puerperium respectively. Subsequently, it rose markedly to

4.39 + 0.49 × 106/μl in late puerperium which was higher than 4.12 + 0.43 ×

106/μl of non-pregnant subjects (Figure VI). Comparing the trend of the mean

34

value of red blood cell count in all groups, it was statistically significant at P

value of 0.001.

WHITE BLOOD CELL COUNT (WBC)

The mean white blood cell count increased gradually form 6.97 + 1.89 ×

103/μl in first trimester to 8.19 + 1.93 × 103/μl in second trimester. It then

dropped to 7.26 + 1.86 × 103/μl in the third trimester (Table II). It thereafter

rose markedly to 10.25 + 3.92 × 103/μl in early puerperium and then declined

sharply to 5.53 + 1.42 × 103/μl in late puerperium which was the lowest value of

all the subgroups (Figure VII). The value for non pregnant subject was 5.82 +

1.62 × 103/μl. Comparing the trend of mean white blood cell count in all groups,

it was statistically significant with the P value of = 0.001.

NEUTROPHIL COUNT

The mean neutrophil count increased from 63.38+7.74% in first trimester

to 69.14 + 6.09% in the second trimester but dropped slightly to 66.72 + 8.25%

in third trimester (Table II). It rose slightly again to 69.71 + 9.10% in early

puerperium but declined markedly to 51.03 + 8.19% in late puerperium close to

the non-pregnant value of 63.38 + 7.74% (Figure VIII). Comparing the trend of

the means of neutrophils in the different groups, the P value of 0.001 showed

statistically significant difference.

35

LYMPHOCYTE COUNT

The mean lymphocyte count decreased from 26.51 + 5.77% in the first

trimester to 22.76 + 6.09% in second trimester. It subsequently increased

slightly to 23.91 + 5.91% in third trimester (Table II). However, it declined to

21.87 + 7.04% in early puerperium and rose markedly thereafter to 37.74 +

8.08% in late puerperium compared to 39.46 + 7.55% in non-pregnant subjects.

(Figure IX). The trend of the mean value of lymphocytes in all groups showed

statistically significant difference at the P value of 0.001.

PLATELET COUNT

The mean platelet count decreased gradually during pregnancy from

239.69 + 62.48 × 103/μl in the first trimester to 214.38 + 54.01 × 103/μl and

202.18 + 55.19 × 103/μl in second and third trimesters respectively (Table II). It

rose slightly to 203.66 + 46.76 × 103/μl in early puerperium and markedly to

245.71 + 58.97 × 103/μl in late puerperium which was close to 250.76 + 69.47

× 103/μl obtained in non-pregnant subjects (Figure X). Six (1.4%) subjects had

platelet count between 94 -100×103/μl and 33(7.8%) had platelet count

between 101-149× 103/μl. Comparing the trend of the means of platelet count in

all groups, the P value of 0.001 showed statistically significant difference.

Comparison of variables across the trimesters of pregnancy and puerperium.

The mean values for each variable in pregnancy were compared between

the trimesters (Table IV). The difference in the mean haematocrit was found to

36

be statistically significant between first and second trimester (P=0.026) and

second and third trimesters (P=0.007). There was no statistically significant

difference in the mean haemoglobin concentration in between trimesters among

the subjects. There was statistically significant difference between RBC count in

first and second trimester only. Other red cell indices MCV, MCH, MCHC showed

no statistically significant difference in between trimesters. Comparison of the

mean values of WBC between first and second trimester showed statistically

significant difference which is similar to that obtained for mean platelet value

between the first and third trimesters.

Table V showed comparison of the mean values among variables in early

puerperium, late puerperium and non-pregnant subjects. The difference in the

mean haematocrit values between i) early and late puerperium (P<0.001), ii)

early puerperium and non-pregnant subjects (P<0.001) and iii) late puerperium

and non-pregnant subjects (P<0.001) were found to be statistically significant.

However, the difference between the mean values of early puerperium and late

puerperium and late puerperium and non-pregnant subjects were found to be

statistically significant in haemoglobin concentration and RBC count values

(P<0.05). No significant difference was observed in MCV, MCH, MCHC when

mean values were compared between early and late puerperium, puerperal and

non-pregnant groups.

The mean values of WBC count also revealed statistically significant

difference between early puerperium and late puerperium (P<0.001). However,

37

only pairing of early puerperium and non-pregnant subjects showed statistically

significant difference in the mean platelet count.

38

TABLES TABLE I Age, Weight, Height and Body Mass Index Values in pregnancy and puerperium First Second Third Early Late Non P-value

Trimester Trimester Trimester Puerperium Puerperium Pregnant

Age 31.04 30.07 30.08 29.99 30.27 29.99 P=0.655

(years) + 3.85 +4.18 +3.85 +4.08 +4.64 +5.68 NS

Weight 69.12 69.06 75.34 73.51 72.68 62.70 P=0.032

(Kg) +14.20 +12.60 +13.80 +10.10 +13.94 +11.60 S*

Height 1.57 1.58 1.58 1.57 1.58 1.59 P=0.867 (m) +1.07 +1.10 +1.40 +1.35 +1.16 +1.22 NS

B.M.I 28.04 27.66 30.20 29.82 29.09 24.80 P=0.089

(Kg/m2) +4.45 +5.80 +4.28 +6.65 +5.56 +3.86 NS

Table Legend: NS=not significant, S*= Statistically significant, B.M.I – body mass index

Values are given as mean + standard deviation

39

TABLE II Haematological values in the three trimesters of pregnancy First Trimester Second Trimester Third trimester Sig. Difference N=70 n=71 n=71

Haematocrit (%) 31.98+3.39 30.24+3.68 32.18+3.19 Yes

(25.50-38.6) (24.9-35.4) (26.4-39.2) p=0.001

Haemoglobin 10.22+0.88 9.63+1.25 10.18+1.14 Yes Concentration (8.00-12.90) (7.80-11.60) (8.80-12.90) p=0.001

(g/dL)

RBC count 3.88+0.28 3.64+0.49 3.84+0.51 Yes

(x106/μl) (3.16-4.63) (2.28-6.14) (2.79-5.38) p=0.001

Mean Corpuscular 82.13+4.84 83.67+5.54 84.41+7.12 No Volume (MCV)(fl) (67.30-93.50) (68.30-96.10) (68.10-105.20) p=0.222

Mean Corpuscular 26.26+2.00 26.55+2.70 26.60+2.91 No Haemoglobin (MCH) (21.10-31.90) (20.50-31.60) (19.70-32.90) p=0.189

(pg)

Mean Corpuscular 32.01+1.54 31.73+1.72 31.59+1.45 Yes Haemoglobin (29.00-36.00) (29.00-35.30) (28.90-35.00) p=0.033

Concentration

(MCHC) (g/dL)

WBC count 6.97+1.89 8.19+1.93 7.26+1.86 Yes (× 103/μl) (3.60-11.10) (4.70-15.40) (3.10-12.40) p=0.001

Neutrophils 63.38+7.74 69.14+6.09 66.72+8.25 Yes

(%) (39.30- 80.20) (54.90-82.50) (48.50-84.50) p=0.001

Lymphocytes 26.52+5.77 22.76+6.09 23.91+5.91 Yes

(%) (11.50-39.40) (8.70-39.90) (11.30-37.90) p=0.001

Platelet count 239.69+62.48 214.38+54.01 202.18+55.19 Yes (× 103/μl) (112 - 439) (102 – 353) (117 – 404) p=0.001

All values = mean + standard deviation, range in parenthesis. Sig.= significant

40

TABLE III Haematological values in puerperium and non-pregnant subjects Early puerperium Late puerperium Non pregnant Sig. Difference n=70 n=70 n=70

Haematorit (%) 31.49+3.71 36.10+3.25 33.84+3.39 Yes

(25.70-37.90) (28.90-48.10) (26.40-39.20) p=0.001

Haemoglobin 10.13+1.28 11.37+1.27 10.50+1.50 Yes Concentration (8.30-12.90) (9.70-14.10) (8.50-15.10) p=0.001

(g/dL)

RBC count 3.86+0.53 4.39+0.49 4.12+0.43 Yes

(x106/μl) (2.75-4.90) (3.33-5.74) (3.10-5.49) p=0.001

Mean Corpuscular 82.86+7.12 82.68+5.94 82.41+5.57 No Volume (MCV)(fl) (67.70-96.50) (67.70-94.20) (64.20-94.10) p=0.222

Mean Corpuscular 26.26+2.00 26.55+2.70 26.60 + 2.91 No Haemoglobin (MCH)pg (20.00-30.40) (21.00-31.30) (18.70-30.20) p=0.189

Mean Corpuscular 32.01 +1.54 31.73 +1.72 31.59 + 1.45 Yes

Haemoglobin (27.10-35.50) (28.60-35.60) (27.90-34.80) p=0.033 Concentration

(MCHC) (g/dL)

WBC count 10.25+3.92 5.53+1.42 5.82+1.62 Yes (× 103/μl) (4.20-23.30) (3.20-9.40) (3.60-13.70) p=0.001

Neutrophils 69.71+9.10 51.03+8.19 48.04+8.32 Yes (%) (46.90- 89.50) (31.20-69.00) (30.90-70.70) p=0.001

Lymphocytes 21.87+7.04 37.74+8.08 39.46+7.55 Yes (%) (7.30-37.80) (22.30-59.40) (22.16-58.00) p=0.001

Platelet count 203.66 +46.76 245.71 +58.97 250.76 +69.47 Yes (× 103/μl) (100 - 317) (117 – 404) (92 – 458) p=0.001

n= number of subjects Sig.= significant

All values are mean + standard deviation, range in parenthesis.

41

TABLE IV

Mean (± S.D) haematological values over the three trimesters of pregnancy in the

study population.

Parameters Trimester 1 Trimester 2 Trimester 3 p-value

1st & 2nd 1st & 3rd 2nd & 3rd

RBC (x106/μl) 3.88 ± 0.28 3.64 ± 0.49 3.84 ± 0.51 0.032* 1.000 0.167

Hb (g/dl) 10.22 ± 0.88 9.63 ± 1.25 10.18 ± 1.14 0.067 1.000 0.116

PCV (%) 31.98 ± 2.31 30.24 ± 3.68 32.18 ± 3.19 0.026* 1.000 0.007*

MCH (pg) 26.26 ± 2.00 26.55 ± 2.70 26.60 ± 2.91 1.000 1.000 1.000

MCHC (g/dl) 32.01 ± 1.54 31.73 ± 1.72 31.59 ± 1.45 1.000 1.000 1.000

MCV (fl) 82.13 ± 4.84 83.67 ± 5.54 84.41 ± 7.10 1.000 0.393 1.000

WBC (× 103/μl) 6.97 ± 1.62 8.19 ± 1.89 7.26 ± 1.86 0.023* 1.000 0.222

Platelets (×

103/μl)

239.69 ±

62.48

214.38 ±

54.01

202.18 ±

55.19 0.152 0.002* 1.000

Table legend text:

RBC – Red blood cell, Hb – Haemoglobin, PCV – Packed cell volume, MCH – Mean corpuscular

haemoglobin, MCHC – Mean corpuscular haemoglobin concentration, MCV – Mean corpuscular

volume, WBC – White blood cell * indicates the level of significance p < 0.05

42

TABLE V

Mean (± S.D) haematological values over the puerperium and non pregnant women

in the study population.

Parameters EPP LPP NP p-value

EPP & LPP EPP& NP LPP & NP

RBC (x106/μl) 3.86 ± 0.53 4.39 ± 0.49 4.12 ± 0.43 0.000* 0.013* 0.011*

Hb (g/dl) 10.13 ± 1.28 11.37 ± 1.27 10.50 ± 1.50 0.000* 1.000 0.001*

PCV (%) 31.49 ± 3.71 36.10 ± 3.25 33.84 ± 3.39 0.000* 0.000* 0.001*

MCH (pg) 26.30 ± 3.13 26.06 ± 2.53 25.52 ± 2.78 1.000 1.000 1.000

MCHC (g/dl) 34.35 ± 1.05 34.42 ± 1.30 34.33 ± 1.43 1.000 0.111 1.000

MCV (fl) 85.89 ± 7.28 87.49 ± 7.02 86.70 ± 6.85 1.000 1.000 1.000

WBC (× 103/μl) 10.25 ± 3.92 5.53 ± 1.42 5.82 ± 1.62 0.000* 0.000* 1.000

Platelets (×

103/μl)

203.55 ±

46.76

245.32 ±

58.97

250.76 ±

69.47 1.000 0.000* 1.000

Table legend text:

RBC – Red blood cell, Hb – Haemoglobin, PCV – Packed cell volume, MCH – Mean corpuscular

haemoglobin, MCHC – Mean corpuscular haemoglobin concentration, MCV – Mean corpuscular

volume, WBC – White blood cell EPP – early puerperium, LPP – late puerperium, NP – non pregnant

women * indicates the level of significance p < 0.05.

43

FIGURES

FIGURE I

Mean Haematocrit (%) in non pregnant state, pregnancy and puerperium

FIGURE II

Mean Haemoglobin concentration (g/dl) in non pregnant state, pregnancy and

puerperium

44

FIGURE III

Mean corpuscular volume (fl) in non pregnant state, pregnancy and puerperium

FIGURE IV

Mean corpuscular haemoglobin (pg) in non pregnant state, pregnancy and

puerperium

45

FIGURE V: Mean corpuscular haemoglobin concentration (g/dl) in non pregnant state,

pregnancy and puerperium

FIGURE VI:

Red Blood Cell count (microlitre) in non pregnant state, pregnancy and puerperium

46

FIGURE VII

White Blood Cell count (microlitre) in non pregnant state, pregnancy and puerperium

FIGURE VIII

Neutrophils (%) in non pregnant state, pregnancy and puerperium

47

FIGURE IX:

Lymphocytes (%) in non pregnant state, pregnancy and puerperium

FIGURE X:

Platelet count ×103/μl) in non pregnant state, pregnancy and puerperium

48

CHAPTER FIVE

DISCUSSION It is well known that pregnancy imposes substantial burden on maternal

haemopoietic system because of the need for erythropoiesis in the face of

expanding blood volume. Therefore studies providing reference haematological

and other parameters in apparently healthy individuals in various physiological

states will certainly continue to be relevant91,92. Although, the phenomenon of

changes in normal laboratory values induced by pregnancy is well recognized,

very few laboratory provide reference range for pregnant women93. This study

discussed the findings of a cross-sectional study of complete blood count utilizing

automated techniques in normal pregnant and puerperal subjects attending a

large tertiary maternity unit in Lagos.

The highest mean weight in the subjects in third trimester of pregnancy is

consistent with the established physiological weight changes in pregnancy since

most weight gain occured in the last two trimesters94. However, despite this

significant difference in weight, significant difference in the body mass index was

not observed in all study groups. This was in agreement with the observation

that the contribution of body mass index to variation in haematological indices

was negligible33.

This study did not demonstrate significant association between parity and

the various study groups. While parity is known to influence haematological

indices in pregnancy95 and puerperium32, the role of parity in determination of

49

haematological profile was not examined in this study. More so,

grandmultiparous women and those with short inter-pregnancy interval which

could lead to depleted iron store with consequent iron deficiency anaemia have

been excluded from this study.

RED BLOOD CELL (RBC) COUNT AND INDICES

This study showed clearly that there is significant fall in haematocrit,

haemoglobin concentration and RBC count in pregnancy especially in the first

and second trimester followed by a small rise in the third trimester. The trend in

these indices is comparable to reports in other studies13,38,96. While some workers

noted consistent decrease in the haematocrit throughout pregnancy39, other did

not find any significant change28,29,31. The mean values of haematocrit in this

study are higher than those reported by Dapper et al31 but similar to that

obtained by Akingbola et al39. The increased haematocrit in the third trimester

has been attributed to plateau of plasma volume expansion from the 30th week

of gestation compared to sustained erythropoiesis throughout pregnancy24,26.

The mean values for haemoglobin concentration in the three pregnant

groups in this study were lower than the 11.0 g/dL recommended by the WHO.

Similar observation has been made in other studies in pregnant Nigerian

women28,29,31,39. By WHO criteria46, 68.2% of this study population presented

with anaemia (52.6% mild and 15.6% moderate anaemia). Similar reports of

50

anaemia in pregnancy have been documented in several Nigerian studies

51.4%95, 50-60%97 and 36-56%98.

However, using a lower haemoglobin concentration of 10g/dL as proposed

by Harrison99 and Ogunbode100, only 15.6% of the subjects would be qualified as

being anaemic. The cause of anemia has been ascribed to increased

physiological demand of pregnancy and plasma volume expansion in excess of

increased red cell mass. The effects are more marked in developing countries

like Nigeria probably due to lack of balanced dietary intake, hookworm

infestation, malaria and frequent pregnancies with short intervals101.

Complications of anaemia include increased risk of miscarriage, still birth,

premature delivery, intra-uterine growth restriction and low birth weight. The

relationship between anaemia and adverse pregnancy outcome, despite much

research, is still unclear. The evidence that maternal anaemia can reduce a

pregnant woman’s ability to withstand blood loss or that it increased the risk of

spontaneous abortion, preterm delivery, low birth weight and maternal

mortality102-104 is inconclusive105,106. However, anaemia once discovered must be

properly evaluated to identify the cause and prompt treatment instituted.

In practice, for more than 3 decades, many hospitals use a lower level of

haemoglobin concentration of 10g/dL or less as indicating anaemia. This level

has been justified on the basis of the work of Lawson in 1967 which showed that

serious harm to the fetus did not occur until haemoglobin value was below

10g/dL or packed cell volume below 30%95. Studies on the relationship between

51

maternal haemoglobin concentration at term and the birth weight did not report

any adverse feto-maternal95 or perinatal outcome107.

It is also noteworthy that hypervolaemia of pregnancy that played a

significant role in occurrence of anaemia in pregnancy is not without benefit.

While it safeguards the mother against adverse effect of blood loss associated

with parturition, it is also important for fetal growth and well-being. This

facilitates adequate blood flow in the feto-placenta unit and enhances transfer of

oxygen and nutrients to the fetus. On the other hand, high haematocrit that may

represent failure of plasma volume expansion can also lead to low birth weight

even after controlling for hypertension and pre-eclampsia108. The mechanism by

which this effect is mediated is unknown but may be related to increased blood

viscosity with consequent disturbance in flow.

In this study, the MCV rose progressively but insignificantly during

pregnancy to the peak in third trimester and gradually decline during the

puerperium close to the non-pregnant level at 6 weeks postpartum. This finding

is similar to reports by other authors31,39,36. This rise in MCV is more pronounced

in iron supplemented pregnant women109 as in this study population. Since the

haemoglobin concentration which provides a quantitative measure of the severity

of anaemia lacks sensitivity and specificity110, the MCV may be a better index of

anaemia in pregnancy than haemoglobin concentration and haematocrit because

both are reduced by increase in plasma volume13.

52

Considering the fact that Lower haematological values has been reported

for Africans than Caucasians2,3, it may be deduced that the apparently lower

values of haematocrit and haemoglobin concentration in the context of normal

MCV and MCHC reflects the true range of normal values of these indices in

Nigerian population rather than being considered anaemic. Similar findings have

been reported by other Nigerian authors28,29,39. For practical use, haemoglobin

concentration (Hb) of 10g/dL or haematocrit of 30% in the context of normal

MCV may be considered as a general discriminatory value for anaemia in

pregnant women in this environment. This has been suggested by previous

workers99,100,112. Therefore incidence of anaemia in the six groups at

haemoglobin concentration of 10g/dL or less becomes 17.6% overall. The

breakdown among different groups was 4%, 20% and 10 % among the first,

second and third trimester groups respectively. While 17%, 3% and 11%

represents anaemia in early puerperium, late puerperium and non-pregnant

groups respectively.

It was also noted in this study that the MCH increased slightly but not

significantly during pregnancy reaching the peak in third trimester. Thereafter, it

declined during puerperium to 26.06pg at 6 weeks post-partum which was close

to the non-pregnant value of 25.52pg. This finding was consistent with similar

reports13,39. Other workers also observed progressive decrease in the MCH during

pregnancy31,96. The MCHC decline slightly but significantly during pregnancy,

then increased marginally in early puerperium and thereafter reduce to

53

31.57g/dL in the late puerperium compared to 31.07g/dL obtained for non-

pregnant subjects. This was consistent with the findings of Taylor and Lind13

The haemoglobin concentration, haematocrit and RBC count dropped

slightly in early puerperium and increased thereafter towards normal level at the

end of puerperium. This finding is comparable to that of other authors13,32,38,96.

While some authors reported similar mean values of haemoglobin and

haematocrit for late puerperium and non-pregnant subjects32,96, the mean values

of these indices in this study at late puerperium were higher than that for non-

pregnant subjects.

The poor correlation between the mean values of all RBCs indices in late

puerperium compared to non-pregnant values suggests that complete return of

these indices to normal may be complex. Taylor and Lind13 in their longitudinal

study did not achieve the non-pregnant values in their subject until 6 months

after delivery. This may be due to the fact that haematological indices do not

undergo simple reversal to non-pregnant values at the end of puerperium

because of probable shift in fluid and cellular compartments which is poorly

understood.

WHITE BLOOD CELLS

The white cell count in this study rose markedly from first to second

trimester and then reduced slightly in the third trimester. It rose again in early

puerperium to the peak level and thereafter decreased significantly during

54

puerperium to reach the non-pregnant value at 6 weeks postpartum. This trend

is similar to that reported by Tameika et al96 but differs from those reports that

noted progressive leucocytosis throughout pregnancy29,39,74. However, few

workers have also reported a decrease31.

The trend in the neutrophil count in this study followed that of white

blood cell supporting the fact that changes in white cell count in pregnancy

predominantly reflects changes in neutrophils112. Many studies have also

reported gradual decrease in WBC and neutrophil count from early to late

puerperium when non-pregnant values were achieved32,38.

The lymphocyte count in this study decreased from first to second

trimester then rose slightly in third trimester to decline again in early

puerperium. Thereafter, it increased significantly during puerperium to a mean

value close to the non-pregnant level at 6 weeks post-partum. This finding was

inconsistent with others that reported gradual decrease in lymphocyte count

during pregnancy38,39. The increased in lymphocyte count during the puerperium

observed in this study was also observed by others workers32,38. The

lymphocytosis in the puerperium has been attributed to presence of soluble

factors (possibly alpha2-globulin and acute phase reactants). Of importance also

is the physiological response to trauma at delivery rather than any reflection of

immuno-regulatory events62,64.

55

THE PLATELET

This study showed that the platelet count decreased progressively during

pregnancy to term. This was the common trend in most reports60,113,114. While

some workers have reported increased platelet count during pregnancy29, it may

follow an undulating pattern by increasing in second and declining in the third

trimester39. The reason behind this fall in platelet count during pregnancy is not

definite. While some workers opined that it may be due to the dilutional effect of

relative increase in plasma volume assuming platelet production is fairly constant

during pregnancy72, others have attributed it to benign gestational

thrombocytopaenia74.

After delivery, the platelet count rose again from early puerperium

markedly to non-pregnant level by 6 weeks postpartum13. The rise in the platelet

count after delivery can be considered as a compensatory increase in platelets

production after a period of platelet consumption during separation and delivery

of the placenta8. This also helps in maintaining haemostasis after delivery to

prevent post-partum haemorrhage.

In this study, 98.6% of all subjects have platelet counts over 100,000

x103/μl while only 1.4% had thrombocytopaenia. This 1.4% value of

platelet count less than 100,000 is lower than the 3.6% reported by Akingbola et

al39. Though, the incidence is low in this study, thrombocytopaenia occurring

56

during pregnancy deserves evaluation. The cause can usually be determined by

thorough history, physical examination and directed laboratory studies.

It is important to consider normal reference ranges specific to pregnancy

when interpreting some laboratory results that may be altered by normal

changes of pregnancy. Unless these normal gestational related alterations are

taken into account when evaluating laboratory values in pregnant and puerperal

women, physiologic adaptations of pregnancy can be misinterpreted as

pathologic or alternatively, pathological findings may not be recognized93.

This study describes the changes in haematological indices in pregnancy

and puerperium and suggests accurate reference range of values for

haematological parameters in apparently normal and healthy pregnant and

puerperal patients. Important observation lies in the fact that while lower

haematological values were reported for pregnant and non-pregnant women,

values of haemoglobin concentration 10g/dL or lower and haematocrit of less

than 30% may be considered as anaemia in presence of normal MCV or MCHC.

57

RECOMMENDATIONS

Conduction of a multi-regional longitudinal survey of

haematological parameters in pregnancy and puerperium should be

undertaken to find out range of values that can be taken as

national average.

Screening of pregnant women for complete blood count in all

trimesters to detect and treat any abnormality that ensue.

Incorporation of pre-conceptional care into the primary health care

programme to screen for and treat women of childbearing age

group for anaemia.

Abnormalities of haematological profile, even with mild clinical

presentation should be thoroughly evaluated.

Administration of haematinics (iron and folic acid supplement) and

malaria chemoprophylaxis to all pregnant women.

58

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CHAPTER SIX APPENDICES

APPENDIX I A PARTICIPANT INFORMED CONSENT FORM (ENGLISH VERSION)

Informed consent for the study to determine the haematological indices of pregnant and postpartum women attending antenatal and postnatal clinics including lying-in ward of the

Department of Obstetrics and Gynaecology, Lagos State University Teaching Hospital, Ikeja, Lagos.

I have been given the following information: 1. That the research is being undertaken to determine the haematological indices of

pregnant and postpartum women attending antenatal and postnatal clinics including

lying-in ward of the Department of Obstetrics and Gynaecology, Lagos State University

Teaching Hospital, Ikeja, Lagos.

2. That I will need to answer some questions about myself.

3. That about 3ml of blood for test will be collected from me.

4. That all precautions will be taken to ensure that the process of examination and blood

letting will not cause me any harm or injury.

5. That my participation in this research will be at no cost to me.

6. That I will not be at any risk for participating in this study.

7. That the information generated from this study will be helpful to Doctors in future to

make better decisions on treatment of pregnant and postpartum women with

derangement in haematological profile.

8. That any abnormality discovered, will be communicated to me.

9. That the information obtained in this study is strictly confidential.

10. That I have the right to ask questions on any aspect of this study that is not clear to me

before I participate.

11. That I am free to refuse to take part in this study if so wish.

12. That I have the right to withdraw at any time during the entire period of the study

13. That my refusal to participate will not in any way affect his professional relationship with

me in the hospital.

I…………………………………………………………………………voluntarily consent to participate in the above named research study being conducted by Dr Oshodi Y.A of Obstetrics and Gynaecology

Department, LASUTH Ikeja.

………………………………………… ………………………………………………………

Date Signature/Thumb print of patient

Note: if you have additional questions later, please contact:

Name: Dr. Oshodi Yusuf Abisowo Dept: Obstetrics and Gynaecology, LASUTH, Ikeja

Mobile: +2348023237168 Email: yusufoshodi@yahoo.com

73

APPENDIX I B

PARTICIPATION INFORMED CONSENT FORM (PIGGIN VERSION)

Dr Oshodi Y.A wey dey work for inside Ayinke House for LASUTH dey carry do one work for insid

di blood of women wey carry belle and dose wey don born pikin to shek him level weda de blood

level dey sam sam. Him wan give u some infomate wey u fit consida weda to join him for di work

or no gree join am.

Dem don give mi di infomate wey dey below:

1. Dat di work wey dem won do na to shek di blood of women wey carry belle and dose

wey don born pikin for inside dis Ayinke House

2. Dat I go nid to follow anser som infomate about meself wit mi oga for house

3. Dat dem go kolekt small blood (3ml) for mi bodi

4. Dat dem go do gidigba say di blood wey dem go tak for mi bodi no go cos mi wahala

5. Dat I no go pay ani moni for dis work wey dem won do

6. Dat I no fit get any damage or wund for my bodi if join insid dis work

7. Dat di infomate wey dem get for dis work go helep doktors for tomorrow tak kia of

woman wey get belle abi born pikin to trit dia blood problem if im no dey well well

8. Dat dem go tell mi if di resut no dey kampe

9. Dat di informate wey I giv go dey for di worku only, nobodi go hia am

10. Dat I fit ask anitin insid di worku wey I no sabi befor I join

11. Dat I fit no join di work if I like

12. Dat I fit commot frum di work any time I like

13. Dat if commot o or no enter join di work at all, e no go spoil di kin way doktor dey take

treat mi for dis hospital

Me ………………………………………….. don gree to join di work wey dem don talk to me about

say Doktor Y.A Oshodi wey dey work for Ayinke House , LASUTH wan do.

………………………………………… ………………………………………………………

Date Signature/Thumb print of patient

Note: If anytin dey wey you was ask, make una fone the number wey dey down:

Name: Dr. Oshodi Yusuf Abisowo Dept: Obstetrics and Gynaecology, LASUTH, Ikeja

Mobile: +2348023237168 Email: yusufoshodi@yahoo.com

74

APPENDIX II

PROFORMA FOR DATA COLLECTION Date: …………

A) Trimester (1st, 2nd or 3rd): ………….. Serial no: ……..

B) Puerperium: Early (2-4 days) ……..Late (6wks): ………. Serial no: ………

C) Non-pregnant: …………………………… Serial no: ………

(For non-pregnant subjects, omit spaces where information is not applicable)

1. Initials: ………………………

2. Age: ……………………….. (b) Hospital No: …………………………………

3. (a) Parity: ……………………. (b). If Multipara, Last Confinement: ……………

4. (a) Occupation: …………………………….. (b). Tribe: …………………

5. (a) Educational Status: ……………………... (b). Religion: ……………...

6. (a) Marital Status: ………………………… (b). Booking status: ………

7. (a) Weight (kg): ……………………………. Height (m) ………………..

8. Last Menstrual Period: a] By date ………… or b] By Early scan …………..

9. Spouse Occupation: ……………………………..

10. Gestational Age: …………………… (wks)

11. (a) Date Delivered: …………………… G.A@ Delivery: ……………………..

12. Estimated Blood loss at Delivery: ………………..

13. Apgar Scores: (a) at 1minute ………….. (b) at 5minutes: ………………..

14. Birth Weight (gm): ………………...

…………………………………

Name of interviewer

75

APPENDIX III

LABORATORY FORM Laboratory No: …………….. Date of Sample collection: ………………. LABORATORY RESULTS: Haematocrit (Hcrt): ………………………………………………………….. % Haemoglobin concentration (Hb): ……………………………………….. g /dl Mean corpuscular volume (MCV): ………………………………………….. fL Mean corpuscular haemoglobin (MCH): ………………………………….. pg Mean corpuscular haemoglobin concentration (MCHC): …………… g/dl

Red Blood Cell count (RBC): ……………………………………….............x106/μl

White Blood Cell count (WBC): ……………………………………………… x103/μl Neutrophils ………………………………………………………………………….. % Lymphocytes: ………………………………………………………………………. %

Platelet count (Platelet): …………………………………………………………x103/μl

……………………………………. …………………………………… Name of Laboratory Technologist Signature and Date