Nigerian Journal of Paediatrics

Coagulation changes in children with sickle cell anaemia during painful crises and steady state at Federal Medical Centre Abeokuta Nigeria

Niger J Paediatr 2018; 45 (4):173 - 179

ORIGINAL

Adebola MB

CC – BY Coagulation changes in children

Olanrewaju DM

Ogundeyi MM

with sickle cell anaemia during

painful crises and steady state at

Federal Medical Centre Abeokuta,

Nigeria

DOI:http://dx.doi.org/10.4314/njp.v45i4.2

Accepted: 3rd December 2018

Abstract :

Background:

The

by sandwich ELISA method using

pathophysiology

vaso-

My Biosource® D-dimer and

Adebola MB (

)

occlusive crises in sickle cell

F1+2 ELISA kits.

Department of Paediatrics,

anaemia (SCA) is multifactorial

Results: Mean D-dimer level was

Babcock University Teaching

and hyper coagulability is be-

7358 ± 4354.33ng/ml in the SCA

Hospital, Ilishan-Remo, Ogun state,

lieved to play a role. The associa-

subjects during painful crises,

Nigeria.

tion between hyper coagulabilty

5509 ± 3506.2ng/ml during steady

Email: mukdijah@yahoo.com

and vaso-occlusive disease has

state, and 800 ± 1874.14ng/ml in

been extensively studied in adult

HbAA controls. Mean (F1+2)

Olanrewaju DM

SCA patients, there is however

level was 0.84± 0.43nmol/l in the

Department of Paediatrics,

paucity of data on the subject re-

SCA subjects during painful crises,

Olabisi Onabanjo University

garding paediatric SCA.

0.64± 0.25nmol/l during steady

Teaching Hospital, Ogun state,

Objective: This study set out to

state, and 0.41 ± 0.28 nmol/l, in

Nigeria.

determine the presence of hyper-

HbAA controls. The mean values

coagulable states specifically in

of both coagulation markers as-

Ogundeyi MM

paediatric SCA subjects through

sayed were significantly higher

Department Paediatrics,

quantification of specific coagula-

during painful crises than at steady

Federal Medical Centre, Abeokuta,

tion markers during painful crises

state (P=0,002), while steady state

Ogun state, Nigeria.

and steady state.

values were also significantly

Methodology: The study was a

higher than that of haemoglobin

hospital-based longitudinal study

AA individuals (P=0.001).

carried out between May and Oc-

Conclusions: This study suggests

tober 2015 at Federal Medical

the presence of hypercoagulable

Center, Abeokuta, Nigeria. Fifty

states in paediatric SCA during

SCA subjects were consecutively

steady state which is exacerbated

recruited during painful crises and

during painful crises. The clinical

followed up into their respective

imports of this finding require fur-

steady states. Twenty-five HbAA

ther elucidation.

individuals served as controls

state. Assays of coagulation mark-

Key words: Sickle cell anaemia,

ers, D-dimer and prothrombin

hypercoagulability, D-dimer.

fragment (F1+2) were carried out

Prothrombin fragment

Introduction

oxygenated HbS molecules to interact with each other to

form the rigid polymers that give RBC the characteristic

sickle shape .Although this pathophysiological scheme

Sickle cell disease (SCD) is an inherited haemolytica-

naemia whose clinical manifestations arise from the

constitutes the basic mechanism of the disease, and ex-

tendency of the haemoglobin S (HbS) to polymerize and

plains the haemolyticanaemia, and the mechanical as-

deform red blood cells into the characteristic sickle

pects of vaso-occlusive crises (VOCs), it does not how-

shape .

ever account for the processes that actually trigger and

perpetuate VOCs .

Acute vaso-occlusive pain is the cardinal feature of

SCA and is largely a consequence of mechanical ob-

struction to blood flow by sickled red cells as well as

More recent findings have implicated RBC dehydration,

increased adhesion of red blood cells (RBC) and leuco-

abnormal RBC adhesion to the endothelium, inflamma-

cytes to the vascular epithelium .

tion, activation of blood cellular elements, abnormalities

of vascular tone and nitric oxide (NO) metabolism, as

The presence of HbS results in a conformational change

well as coagulation activation in the pathogenesis of

VOCs.

6-10

in the haemoglobin tetramer which causes the de-

174

In spite of recent improvements in management, mor-

state, as well as neighbouring South-western states of

bidity and mortality from SCA remain high in the West

Nigeria.

African sub-region. . Painful crises are the hallmarks of

The hospital’s services include a weekly paediatri-

SCA, and coagulation activation is now believed to play

chaematology clinic which has over 330 registered SCD

a role in the development of these crises

12, 13

subjects. The clinic holds every Tuesday with an aver-

Previous studies

12-15

have established the existence of

age weekly attendance of 20 SCA patients, and is run by

hypercoagulable states with elevated fibrin D-dimer,

a Consultant Paediatric Haematologist assisted by one

prothrombin fragments and thrombin-antithrombin com-

senior and junior resident each, as well as an intern. The

plex levels during steady state in SCA subjects which

paediatric Unit also comprises of the Children’s emer-

are exacerbated during painful crises and other compli-

gency room, the children’s wards, the newborn unit as

cations of SCA. Many of these studies

12-14

have, how-

well as the paediatric general out-patient department.

ever, beenlargely restricted to adult populations. Stud-

ies

16-18

have, interestingly, also established elevated lev-

Study population

els of specific coagulation markers in relation to aging

and smoking even in the absence of overt clinical car-

Confirmed SCA subjects who presented with painful

diovascular disease. These latter findings limit the ex-

crises at the children emergecy room or the haematology

trapolation of findings of elevated specific coagulation

clinic and met the inclusion criteria were recruited for

markers in adults SCA patients to paediatric populatons.

the study. These were confirmed SCA patients aged

Furthermore, there is paucity of data on coagulation

between 3-15years who presented with painful crises at

changes in paediatric SCA patients from the West Afri-

the children emergency room or haematology clinic at

can sub-region. Only one study

from the sub-region

FMC Abeokuta. Three years is the minimum age for

which assayed Fibrin D-dimer in adult SCA subjects in

which the Oucher pain chart has been validated for ap-

Ibadan was available to this investigator; while none

plication, while 15 years is the cut-off age for paediatric

assaying more than one specific coagulation marker

care at the FMC Abeokuta. Painful crisis was defined as

specifically in paediatric SCA patients has been reported

acute painful episodes in SCA subjects not attributable

to any specific aetiology

21, 22

from the Sub-region.

An Enugu

study also examined clotting profiles of

Subjects were consecutively recruited over a period of

paediatric SCA subjects and demonstrated significantly

six months till the estimated sample size of fifty was

prolonged PT and PTTK values during steady state and

reached. The same patients were followed up into their

painful crises. This finding however suggests a predis-

respective steady states which was taken as six weeks

position to abnormal bleeding rather than thrombosis .

after resolution of painful crises or infections, and three

months after the last blood transfusion.

Furthermore, coagulation factors are known to normally

The consents

circulate as inactive zymogens in significant concentra-

of care-givers were obtained, as well as assents of chil-

tions. Elevated levels of clotting factors alone therefore

dren aged 7years and above.

hypercoagulability , which is more

do not confirm

reliably determined through direct measurement of

SCA subjects with painful episodes with determined

markers of thrombin generation such as D-dimer,

aetiologies, established disorders associated with hyper-

prothrombin

fragment(F1+@2)

and

thrombin-

coagulability in children (specifically nephrotic syn-

antithrombin complex (TAT) .

drome and diabetes mellitus were screened for), other

SCD variants obtained by Hb electrophoresis, as well as

This study therefore set out to determine the presence of

those who had been commenced on hydroxyurea, were

a hypercoagulable state specifically in children with

all excluded from the study.

SCA during painful crises and steady state, through as-

say of specific coagulation markers, fibrin D-dimer and

Twenty-five age and sex matched HbAA patients served

F1+2. It also seeks to examine the relationship between

as controls. These were apparently healthy individuals

levels of these coagulation markers and objective assess-

visiting the out-patient clinic for routine follow-up or

ment of pain in subjects.

pre-school entry examination.

The sample size was calculated using the Leslie Fischer

formula. with level of precision set at 4%, and an an-

Methodology

ticipated attrition rate of 10%

The study was a hospital-based longitudinal study car-

Ethical approval for the study was obtained from the

ried out at Federal Medical Centre Abeokuta between

institution’s Research/Ethics Committee . Voluntarily

May and October 2016.

signed informed consent was obtained from the parents

of all subjects and controls. Assents of children above

Study setting

seven years were also obtained. Confidentiality was

maintained by allotting a serial number to each partici-

The study was carried out at the Federal Medical Center,

pant by which they were referenced at all stages of the

Abeokuta, a 250- bedded multi-specialist hospital which

study.

provides tertiary healthcare for inhabitants of Ogun

The sample collection process resulted in fleeting pain at

175

six months

25, 26

puncture sites. Blood samples obtained specifically for

. D-Dimer and F1+2 values were deter-

the purpose of this study were processed at no cost to

mined from these stored plasma samples by sandwich

the patient, and the management of those patients who

ELISA immunoassay using MyBiosource® ELISA kits

were unwilling to participate was not compromised by

The 1ml EDTA venous blood sample was used for

their non-participation..

haemotogical profiles of subjects and controls using an

automated haematologyanalyser.

Materials

Spot urine samples obtained from subjects and controls

were analyzed for proteinuria, glycosuria and ketonuria,

The Oucher Pain Chart

was utilized to assign pain

to screen for diabetes mellitus and nephrotic syndrome.

scores to subjects during painful crisis. This is a poster

These are two easily detectable disorders known to pre-

developed to help children communicate how much pain

dispose to hypercoagulability in children. Patients with

they feel. A picture matching the perceived degree of

spot urinary protein ≥3+ (≥300mg/dl) were to be evalu-

pain was selected from the chart and a corresponding

ated for nephrotic syndrome and excluded, while pa-

score on a scale of 0-10 assigned. Caucasian, Hispanic,

tients with glycosuria were to be further evaluated for

Asian and African-American versions of the chart have

diabetes mellitus and those with random blood glucose

been developed. The African-American version of the

values ≥ 200mg/dl excluded .

chart was employed for this study.

Data management

D-dimer and F1+2 assays were carried out using MY-

BIOSOURCER® Human D-DIMER ELISA Kit

Pre-tested proforma were administered to subjects by

(Catalogue

number:

MBS723523)

and

MYBIO-

interview method by the investigator and two trained

SOURCE® Human Prothrombin Fragment 1+2 ELISA

assistants who were junior residents in the paediatrics

Kits (Catalogue number: MBS701341) respectively.

department. Pain scores were assigned to patients in

Platelet counts were determined using Sysmex 10001®

painful crisis using the Oucher chart and scores re-

Automated Haematology Analyzer.

corded.

D-dimer Reference value: ≤ 250ng/ml. 27

F1+2 Reference value : <0.3nmol/L.

Data analysis

Data were analyzed using Statistical Package for Social

Methods

Sciences (SPSS), version 20.0.

The significance of the differences between mean values

Five and one ml venous blood samples each were col-

expressed in contingency tables was tested using the

lected into plastic EDTA sample bottles by venipuncture

paired sample t-test with level of significance set at p-

from 50 consecutive SCA subjects during painful crises.

value ≤0.05.

Tourniquets were avoided as much as possible during

Pearson correlation coefficient was used to determine

sample collection. Tourniquet use, particularly when

associations between D-dimer and F1+2 values at 95%

prolonged beyond 1 minute, is known to result in activa-

confidence interval, as well as the associations between

tion of coagulation factors and therefore may cause arte-

both coagulation markers and the pain scores of the sub-

factual changes in coagulation profiles

Pain scores

jects.

were respectively assigned to each subject at the point of

recruitment using the Oucher faces pain chart

The venous samples for full blood count and the spe-

cific coagulation markers were obtained from partici-

Results

pants within 72 hours of onset of painful crises. Similar

Clinical variables of subjects and controls

steady state bloodsamples were obtained from the same

patients at least 6 weeks after resolution of painful cri-

A total of 50 SCA patients were recruited for the study

ses,or 3 months afterwards in those transfused during

consisting of 35 (70.0%) males and 15 (30.0%) females.

painful episodes.

Male-female ratio was 2.3:1. The control group con-

sisted of 25 closely matched HbAA individuals, 15

Venous blood samples for full blood count and specific

(60.0%) of which were males and 10 (40.0%) females,

coagulation markers were also obtained from 25 control

giving a male-female ratio of 1.5:1 (Table-1).

subjects who were apparently healthy closely matched

The mean pain score using the Oucher pain chart was

HbAA subjects drawn from the Paediatrics Out-Patient

5.07(±2.2). Using the descriptive pain scale of mild

Department. This group also had their haemoglobin

(pain scores 1-3), moderate (4-6) and severe (7-10), ma-

genotypes determined by electrophoresis using cellulose

jority of the subjects (52.2%) presented with moderate

acetate strips.

pain, 26.1% with mild pain and 21.7% severe pain.

The 5ml venous blood samples for coagulation marker

Laboratory variables of subjects and controls

assays were centrifuged at 3000 revolutions/min, and

the platelet-poor plasma obtained aliquoted into plastic

D-dimer values ranged from 840 to 15660ng/ml (mean:

plain sample bottles and stored at -80°C. Plasma sam-

7358± 4354.33ng/ml) in the SCA subjects during pain-

ples stored at this temperature remain viable for up to

ful crises, 320 to 13200ng/ml (mean: 5509 ± 3506.3ng/

176

ml) during steady state, and 90 to 8960ng/ml

Comparison of laboratory variables of subjects and con-

(800±1874.14ng/ml) in HbAA controls. D-dimer value

trols

above 250ng/L is elevated and suggestive of on-going

thrombosis.

The levels of both coagulation markers were signifi-

cantly higher (D-dimer p= 0.02; F1+2 P= 0.01) during

F1+2

values

ranged

from

0.06

1.81nmol/L

painful crises than in steady state amongst the SCA sub-

( 0.84±0.43nmol/L) in the SCA subjects during painful

jects. There was however no significant difference be-

crises, 0.07 to 1.11nmol/l (0.64±0.25nmol/L) during

tween crisis and steady state platelets in the SCA sub-

steady state, and 0.05 to 1.21nmol/l (0.41±0.28nmol/L)

jects. The steady state levels of both coagulation mark-

in HbAA controls. F1+2 values above 0.3nmol/L are

ers, as well as platelet counts, were also significantly

elevated.

higher than those of HbAA individuals (d-dimer P=

The mean platelet count was 297 X 10 /L during painful

0.00; F1+2 P=0.01) .Mean d-dimer level during painful

crisis, 306 X 10 /L during steady state, and 235 X 10 /L

crises was about ten-fold that of the HbAA controls,

in HbAA controls.

whereas mean painful crises level of prothrombin frag-

ment doubled that of HbAA controls.

There was, however, no significant correlation between

Table 1: Control subjects with unexplained elevated coagula-

grade of pain and both D-dimer and prothrombin frag-

tion marker levels

ment levels (r= 0.160; p= 0.228 and r=0.116; p= 0.453

Subject

D-dimer ( ng/L)

F1+2 (nmol/L)

respectively). Likewise, there was no significant correla-

Ref: <250ng/L

Ref: <0.3nmol/L

tion between d-dimer and prothrombin fragment levels

Control 12

8960

1.86

in SCA patients during painful crises (r= -0.063; p=

Control 14

3920

1.25

0.673).

Control 15

1330

0.95

Table 4: comparison of mean values of coagulation variable

for sca patients in crisis and normal hemoglobin aa children

Table 2: Comparison of mean values of coagulation

Coagulation

HbSS (n=50)

HbAA

variables for SCA patients in steady state and vaso-

markers

Vaso-occlusive

(n=25)

occlusive crisis

crisis

Control

Coagulation

HbSS (n=50)

mean±SD

Markers

Vaso-occlusive

Steady State

D-Dimer(ng/

7358(4254.33)

800

7,34

0.00*

crisis mean±SD

mean±SD

(1874.14)

D-Dimer(ng/L)

7358(4354.33)

5509(3506.21)

2.34

0.02*

Prothrombin

0.83(0.43)

0.41(0.28)

4.43

0.00*

Prothrombin

0.83(0.43)

0.64(0.25)

2.70

0.01*

fragments

(nmol/L)

Platelet count

297(123.48)

306(119.61)

0.37

0.70

(x 10 /l)

*P-values ≤0.05 are significant

Mean D-dimer value during painful crisis was about tenfold

that of HbAA controls while the mean prothrombin fragment

value during painful crisis was about two times that of HbAA

There was a significant difference between mean values

subjects. The differences between these mean values were both

of both D-Dimer and prothrombin fragment in SCA sub-

statistically significant

jects during painful crisis compared with steady state

Fig 1: Scatter graph showing correlation between grade of

Table 3: Comparison of mean values of coagulation variable

pain and D-dimer levels in sickle cell crisis state (r=0.160; p =

for steady state SCA patients and normal hemoglobin aa chil-

0.228).

dren

Coagulation

HbSS

HbAA

Markers

(n=50)

(n=25)

Steady state

Control

mean±SD

D-Dimer(ng/

5509.0

800

6.27

0.00

(3506.21)

(1874.14)

Prothrombin

0.64(0.25)

0.41(0.28)

3.61

0.01

fragments

(nmol/L)

Platelet count

306 (119.6)

235 (92,81)

2,60

0.01

( x 10 /L)

*P-values ≤0.05 are significant

The graph shows that there was no significant correlation

There was a significant difference between mean values of

between D-dimer level during painful crisis and the pain

both D-dimer and prothrombin fragment during steady state

scores of subjects in this study

compared with those of HbAA controls

177

Fig 2: Scatter graph showing correlation between pain grade

between the values of these markers in SCA subjects

and Prothrombin fragment levels in sickle cell crisis state

both during painful crises and steady state compared to

(r=0.116; p= 0.453).

the HbAA controls.

It should be noted also that 3 of the control subjects had

considerably elevated levels of both coagulation markers

which contributed to the high mean values obtained for

the control group (Table1). These are suspected to be

artefactual, possibly due to difficult sampling. Although

we did set out to specifically exclude nephrotic syn-

drome and diabetes mellitus, being two common child-

hood disorders that predispose to hypercoagulaility in

children, we however did not evaluate subjects for levels

anti-coagulant proteins (C and S). Therefore we

could not conclusively exclude the possibility of inher-

ited or acquired abnormalities of these proteins being

responsible for the elevated levels observed in these

The graph shows that there was no significant correlation

HbAA controls.

between prothrombin fragment level during painful crisis and

the pain scores of subjects.

Our findings are in agreement with previous studies in

adult SCA subjects

12-14

which have documented simi-

Fig 3: Scatter graph showing correlation between D-dimer and

larobservations from assays of specific coagulation

Prothrombin fragment levels in sickle cell crisis state (r= -

0.063;p= 0.673).

markers either singly or in various combinations. These

findings have however not been extrapolated to paediat-

ric sickle cell anaemia subjects. Elevated levels of co-

agulation markers have also been well documented

16-18

in apparently healthy adults in relation to aging, as well

as in association with smoking even in the absence of

overt clinical cardiovascular disease.

Both D-dimer and prothrombin fragment are specific

coagulation markers, significant plasma levels of which

represent sufficient evidence of in vivo thrombin genera-

tion

28,30

Patients with SCA exhibit high plasma levels of

markers of thrombin generation; depletion of circulating

anticoagulants protein C and S, increased tissue factor

expression, as well as activation of platelets and other

cellular elements which are chronically activated even in

non-crises states.

There was no significant correlation between D-dimer and

prothrombin fragment levels in this study.

This study also demonstrated significantly higher mean

absolute platelet counts in subjects during steady state

compared to HbAA controls (though the mean platelet

Discussion

counts were within normal limits in the three groups).

This was similar to findings in a Lagos study.

12-14

Previous studies

have demonstrated evidence of

Chronic circulating platelets activation is known to oc-

thrombin generation in association with virtually all

cur in SCA, and contributes to the hypercoagulable state

complications of SCA in adults. Given the heterogene-

in this disorder. Activated platelets release agonists such

ity of presenting symptoms in children with SCA, how-

as adenosine diphosphste (ADP),

adenosine triphos-

ever, this study restricted itself to painful crises as a

phate (ADP), calcium, serotinin and coagulation factors

common denominator across the recruited subjects.

into the surrounding milieu. In addition, platelets also

This study demonstrated a significant increase in D-

provide the catalytic surface for he sequential activation

dimer and F1+2 levels in steady state compared to

of coagulation factors which culminates in thrombin

generation . Decreased bio-availability of is

closely matched HbAA individuals, with a significant

NO, a

further increase during painful crises above steady state

feature of SCD, is also known to contribute to platelet

activation in SCD .

levels. This finding is indicative of on-going thrombin

generation which is suficient evident of coagulation acti-

vation in SCA in children during steady state with a fur-

This study however did not demonstrate a significant

ther exacerbation during painful crises.

correlation between D-dimer and prothrombin fragment

Although mean values for both coagulation markers

levels during painful crises. The time lapse following

were also above reference values in the HbAA controls,

onset of painful crises may explain the insignificant cor-

coagulation .

we demonstrated a statistically significant difference

relation

between

the

markers

178

Prothrombin is released during thrombin generation,

anaemia in children. It is postulated that amelioration of

while fibrin D-dimer is a product of fibrinolysis . It

vascular occlusion pharmacologically would possibly be

should be noted that samples for both coagulation mark-

stronger evidence that the hypercoagulability contributes

to vaso-occlusive complications of SCD . Current prac-

ers were obtained at the same point during crises and

steady state respectively in this study. Although no spe-

tice is however largely empirical, and existing evidence

cific time frame has been suggested for estimation of

on the benefits of these proposed interventions inconclu-

sive

8, 33-35

various coagulation markers in hypercoagulable states, a

previous study

postulated that prothrombin fragment

may be a more useful marker for the earlier phase of

thrombosis whereas D-dimer reflects fibrinolysis after

clot formation. The same study also reported that

Conclusion

Prothrombin fragment was observed to correlate better

with thrombin-anti-thrombin (TAT) complex than with

D-dimer. TAT was however not assayed in this study.

This study indicates the presence of a hypercoagulable

A significant correlation was not observed between pain

state in children with SCA during steady

scores and the levels of D-dimer and prothrombin frag-

state which is further exacerbated during painful crises.

ment in the present study. This corroborates findings

There was no significant association between levels of

from a similar study in adults from Ibadan. Differences

coagulation markers and severity of painful episodes in

in individual pain thresholds make pain assessment a

paediatric SCA subjects. The clinical implications of

subjective clinical end point and may explain the insig-

hypercoagulability in management of painful crises, as

nificant correlation between pain scores and levels of

well as other complications of SCA, however require

specific coagulation markers in this study. Development

further investigations.

of painful crises is also an interplay between several

variables. Apart from the cellular mechanisms known to

contribute to development of VOC, other extraneous

Conflict of interest: None

factors such as extremes of temperatures, dehydration

Funding: None

and infections also play a role. The precise levels of

coagulation markers detectable at any point in time is

also dependent on time elapsed since triggering of

thrombin generation as well as the rate of fibrinolysis.

Limitation

Some studies

7, 12

have reported that D-dimer levels cor-

relate with the frequency of pain episodes measured

This study was limited in not assaying for anti-

during the following year as well as the interval for de-

coagulation proteins (C and S), deficiencies of which are

velopment of pain episodes in adult SCA subjects rather

known to contribute to hypercoagulability in SCD. This

than the severity of painful episodes.

is particularly of importance in the light of the unex-

plained elevated levels of coagulation markers found in

a number of the HbAA control subjects.

The findings from the present study suggest the presence

of a hypercoagulable state in children with SCA detect-

Recommendations

able through assay of specific markers of coagulation, D

-dimer and prothrombin fragment. The clinical imports

Multi-centre studies are required to further elucidate the

of these findings however require further exploration

role of hypercoagulability in complications of SCA, and

and may chart a new course in the management of pain-

possibly chart a new course in case management.

ful crises as well as other complications of sickle cell

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