Haematological values in homozygous sickle cell disease in steady state and haemoglobin phenotypes AA controls in Lagos, Nigeria
BMC Research Notes
Haematological values in homozygous sickle cell disease in steady state and haemoglobin phenotypes AA controls in Lagos, Nigeria
Akinsegun Akinbami 0
Adedoyin Dosunmu 0
Adewumi Adediran 1
Olajumoke Oshinaike 2
Phillip Adebola 2
Olanrewaju Arogundade 0
0 Department of Haematology and Blood Transfusion, Lagos State University, College of Medicine , Lagos , Nigeria
1 Department of Haematology and Blood Transfusion, Faculty of Clinical Sciences, College of Medicine, University of Lagos , Lagos , Nigeria
2 Department of Medicine, Lagos State University, College of Medicine , Lagos , Nigeria
Background: Sickle cell disease is a genetic abnormality involving the haemoglobin. Although, it is primarily a red cell disorders, the white blood cells and platelets are also affected by the mutation. The consequent haemoglobin S causes polymerization of haemoglobin resulting in haemolysis and anaemia. This study aims to provide baseline haematological values in sickle cell disease patients in steady state and compare the deviation from haemoglobin phenotype AA control values. Methods: A case-control study was conducted amongst homozygous sickle cell patients attending the sickle cell clinics of Lagos State University Teaching Hospital Ikeja and haemoglobin phenotype AA controls. About 4.5mls of blood sample was collected from each participant for full blood count analysis. All blood samples were screened for HIV and haemoglobin phenotypes confirmed using cellulose acetate haemoglobin electrophoresis at pH 8.6. Conclusion: Homozygous sickle cell disease patients have lower values of red cell parameters, but higher values of white cell and platelets counts compared to haemoglobin phenotype AA controls.
Haematological values; Homozygous sickle cell disease; Steady state; Haemoglobin phenotype AA
Sickle cell disease is a genetic abnormality involving the
haemoglobin. Patients present with a wide spectrum of
disorders because of a single-point mutation in which
thymine substitute for adenine, thereby encoding valine
instead of glutamine in the sixth position of the
betachain. Haemoglobin S resulting from the substitution
causes polymerization of haemoglobin and red cell
sickling on exposure to low oxygen tension and unsickle on
The repeated sickling and unsickling damages the red
cell membrane leading to irreversibly sickled red cell
even when the oxygen pressure is increased thus
reducing red cell life span as a result of membrane damage
inducing anaemia. The white blood cells and platelets
are also affected by the mutation.
Red blood cells in sickle cell disease
Quantitative and qualitative changes in red blood cells
have been reported. Haemolysis consequent to the
damaged red cell membrane could be intravascular or
extravascular. The former results from the lysis of
complement-sensitive red cells  and haemoglobin lost
during sickling-induced membrane damaged. [2,3] The
latter, occurs by phagocytosis of red cells that have
undergone sickling [4,5] and physical entrapment of
rheologically compromised red cells. Increased
susceptibility to mechanically induced cell fragmentation
has been documented in-vitro and in sickle cell patients
undergoing vigorous exercise .
Degree of haemolysis is inversely related to
haemoglobin concentration and packed cell volume in sickle cell
anemia patient . Numerous factors affect haemolysis
in sickle cell anaemia, percentage of irreversible sickle
cell is of greatest significance.  The degree of
haemoglobin polymer formation, calculated from the mean
corpuscular haemoglobin concentration and the relative
proportion of haemoglobin fractions also correlates
closely with the severity of haemolysis. [8,9].
White blood cells in sickle cell disease
Although sickle cell disease is primarily a disease of the
red blood cell, leucocytes, because of their sizes obstruct
blood vessels more effectively than red blood cells when
attached to the endothelium. The red blood cells
measures 7.2 μm, while small lymphocytes measures 10 μm,
neutrophils 10–14 μm , large lymphocytes 12–16 μm,
monocytes 14-20 μm. Bacterial infection associated with
leucocytosis is a known predisposing factor to sickle cell
disease crises [10,11]. A high absolute neutrophil count
showed statistically significant relationship with clinical
severity of sickle cell anaemia .
Many complications of sickle cell disease are
associated with leucocytosis. It is a risk factor for early sickle
cell disease –related death.  It is implicated in
clinically overt stroke [14,15].
Pathogenesis of silent cerebral infarction  and
acute chest syndrome  have been associated with
Platelets in sickle cell disease
Unlike the red and white blood cells, the clinical effects
of platelets on sickle cell disease are not well established
. However, an association between stroke in sickle
cell disease and platelet count > 450,000/μl has been
reported . Qualitatively, poor platelet aggregation
responses to epinephrine and ADP were also reported in
sickle cell disease .
The objective of this study was to provide baseline
haematological values in sickle cell anaemia patients in
steady state and compare deviation of these parameters
from normal controls with haemoglobin phenotypes AA.
A case–control study was conducted amongst Sickle cell
patients in steady state attending the sickle cell clinics of
Lagos State University Teaching Hospital Ikeja (LASUTH) referred from private clinics outside the
hospital and other clinics in the hospital and whose
diagnoses were confirmed by alkaline haemoglobin
electrophoresis and controls consisting of medical students,
doctors and nurses of the institution between September
to December 2011 after obtaining approval from
LASUTH, the institution’s Ethics and Research
Committee. Written and verbal consents were obtained from
Sickle cell patients filled validated structured
questionnaires including demographic information, history of
previous blood transfusion, and surgery, previous history
of crises, date of last crises, cigarettes and alcohol intake.
Inclusion criteria were; patients with haemoglobin
phenotype SS, no history of crises in the past 3 months
established by a careful history and complete physical
examination, no previous history of surgery, no history
of blood transfusion in the past 3 months. Exclusion
criteria were; history of blood transfusion in the past
3 months, haemoglobin phenotype SC patients, previous
history of surgery, and HIV infected patients.
All controls were phenotype AA as confirmed by Hb
electrophoresis and were asked to fill questionnaires
containing demographic information. Controls with
haemoglobin phenotype AS or SC were excluded from
the study. All patients were on routine tablets used in
the sickle cell clinics i.e. folic acid ,paludrine and vitamin
B complex tablets, none of them was on hydroxyurea
therapy which could impact on the reported results for
the patient group.
Collection of samples
A blood sample of 4.5mls was collected from both
patients and controls into Ethylene Diamene Tetraacetic
acid (EDTA) anticoagulant bottle for full blood count
analysis done on the same day of collection using
Sysmex KN-21 N,(manufactured by Sysmex corporation
Kobe, Japan) a three- part auto analyzer able to run 19
parameters per sample including haemoglobin
concentration, packed cell volume, red blood cell concentration,
mean corpuscular haemoglobin, mean cell volume, mean
corpuscular haemoglobin concentration, white blood
cells and platelet parameters.
Well mixed blood sample was aspirated, by letting the
equipment sampling probe into the blood sample and
then pressing the start button. Approx. 20ul of blood
was aspirated by the auto analyzer. Result of analysis is
displayed after about 30secs.A printout copy of result is
released on the thermal printing paper.
All blood samples were also screened for HIV using
determine rapid kit , and haemoglobin phenotypes
of all patients and controls were confirmed using
cellulose acetate haemoglobin electrophoresis at pH 8.6.
About 50 ul of EDTA blood samples of patients and
controls were lysed with equal volume of water and
haemoglobin separated out with carbon tetrachloride.
Lysed samples of patients and controls and known AA,
AS, SC, and SS controls were placed on cellulose acetate
paper in batches using an applicator . The paper was
placed in electrophoretic tank containing tris-buffer, pH
8.6,electric current was applied. All samples and AA,
AS, SC, and SS controls samples migrated from negative
to positive pole at 400volt. Haemoglobin separation
occurs within 4-6minutes, haemoglobin bands were
compared with the known controls.
Data were analyzed using SPSS version 16.0 (Statistical
Package for Social Sciences, Inc., Chicago, Ill). The
descriptive data were given as means ± standard deviation
(SD. The Pearson chi squared test was used for analytic
assessment and the differences were considered to be
statistically significant when the p value obtained was < 0.05.
Demographic characteristics of cases and controls
A total of 103 cases and 98 controls were enrolled. Cases
consisted of 57 (55.33%) females and 46 (44.66%) males,
while controls were made up of 68 (69.38%) females and
30 (30.61%) males (Table 1).
Mean age ± SD 22.62 ± 6.93 24.72 ± 8.37 28.74 ± 9.47 31.18 ± 10.49
The overall mean age of the cases was
23.79 ± 7.81 years, and control was 30.43 ± 10.19 years.
The minimum age of cases was 14 and maximum of
44 years and for controls were 17 years and 57 years
respectively. All participants tested negative to HIV
Hematological indices in study population
For cases, the overall mean haemoglobin concentration
was 7.93 ± 1.47 g/dl, packed cell volume 24.44 ± 4.68%,
mean cell volume 81.52 ± 7.89 fl, mean cell haemoglobin
26.50 ± 3.20 pg, and mean cell haemoglobin
concentration 32.50 ± 1.07 g/dl. While for controls, mean
haemoglobin concentration was 13.83 ± 1.32 g/dl, packed cell
volume 43.07 ± 3.95%, mean cell volume 86.90 ± 4.69 fl,
mean cell haemoglobin 28.50 ± 1.34 pg, and mean cell
haemoglobin concentration 32.06 ± 0.90 g/dl (Table 2).
The mean haemoglobin concentration of the male cases
was 8.11 ± 1.53 g/dl, packed cell volume 25.04 ± 4.90%,
mean cell volume 81.71 ± 6.34 fl, mean cell haemoglobin
26.54 ± 2.46 pg, mean cell haemoglobin concentration
32.45 ± 1.00 g/dl, and male controls mean haemoglobin
concentration was13.83 ± 1.32 g/dl, packed cell volume
43.07 ± 3.95%, mean cell volume 86.90 ± 4.69 fl, mean cell
haemoglobin 28.25 ± 1.34 pg and mean cell haemoglobin
concentration 32.06 ± 0.90 g/dl. For the female cases the
mean haemoglobin concentration was 7.78 ± 1.42 g/dl,
packed cell volume 23.95 ± 4.49% , mean cell volume
81.36 ± 9.01 fl, mean cell haemoglobin 26.47 ± 3.71 pg ,
mean cell haemoglobin concentration 32.54 ± 1.08 g/dl.
For female controls mean haemoglobin concentration
was11.96 ± 3.10 g/dl, packed cell volume 36.59 ± 3.30%,
mean cell volume 84.63 ± 8.82 fl, mean cell haemoglobin
27.20 ± 1.92 pg, mean cell haemoglobin concentration
31.59 ± 0.93 g/dl (Table 2).
Majority of the cases 62 of 103 (60.2%) had packed cell
volume between 20-30%, followed by 29 of 103 (28.2%)
who had values less than 20% and only 12 of 103
(11.7%) had values greater than 30%. Almost all the
controls 97 of 98 (99%) had packed cell volume greater than
30%. While only 1 of 98 (1%) had a value less than 30%.
For the cases, the overall mean white blood cell counts
was 10.27 ± 3.94 *103/μl and platelet counts of
412.71 ± 145.09*103/μl. While the overall mean white
blood cell count for the controls was 5.67 ± 1.59*103/μl
and platelet counts of 222.82 ± 57.62*103/μl. The mean
white blood cell for males cases was 10.82 ± 4.95*103/μl
and platelet counts 408.40 ± 133.42*103/μl and the mean
white blood cell count for females cases was 9.83 ± 2.86
*103/μl and platelet counts of 416.12 ± 155.09*103/μl.
The mean white blood cell count for males controls was
5.75 ± 1.63*103/μl, and platelet counts of 239.00 ± 62.25
and the mean white blood cell count for females
23.79 ± 7.81 30.43 ± 10.19
Table 2 Mean values of full blood count parameters
Abbreviations: Hgb—haemoglobin concentration, PCV--Packed cell volume, MCV-Mean cell volume, MCH—Mean cell haemoglobin, MCHC-Mean cell haemoglobin
concentration, WBC-White blood cells*109/l, Plts-Platelets*109/l.
controls was 5.63 ± 1.59; and platelet counts of
222.15 ± 58.03 (Table 2).
Most of the cases 71 of 103 (68.9%) had white blood
cell count less than 12,000/μl but a substantial number,
32 of 103 (31.1%) had white blood cell count more than
12,000/μl. Only 1 of 98 controls (1%) had white blood
cell count more than 12,000/μl. Cross tabulating white
blood cell count with frequency of bone pain showed no
statistically significant relationship p value = 0.322.
Almost half of the cases 43 of 103 (41.8%) had platelet
count greater than 450,000/μl, while 60 of 103 (58.3%)
had platelet count less than 450,000/μl. None of the
controls had platelet count greater than 450,000/μl. A
statistically significant relationship was found between
platelet counts and packed cell volume of the subjects
p = 0.001 (Table 3).
This study highlights haematologic reference ranges of
homozygous sickle cell patients compared with normal
controls with haemoglobin phenotypes AA. The red cell
indices were generally lower in sickle cell patients than
controls with haemoglobin phenotype AA while the
white blood cells and platelet counts were higher than
control values. These results were expected considering
the degree of chronic haemolysis, higher risk of
infections and chronic pain in sickle cell patients. Most
Table 3 Bivariate analysis of degree of anaemia with
platelet counts in cases
High Platelets > Normal Platelets Total P value
450,000/μl < 450,000/μl
Abbreviation. PCV-Packed cell volume.
patients have adapted to low red cell indices; there is
therefore no clinical benefit to treat anaemia with blood
transfusion. On the contrary, raising the packed cell
volume to over 30% could increase blood viscosity, which
increases with high packed cell volume .
Pain may be responsible for the leucocytosis seen in
sickle cell anaemia. The overall mean white blood cells
count of 10.27 ± 3.94*103/μl amongst homozygous sickle
cell disease patients doubles value obtained in HbAA
controls 5.67 ± 1.59*103/μl. Due to re-distribution of the
white cells between the marginal and circulating pools,
pain, nausea and vomiting and anxiety have been
reported to cause leucocytosis in the absence of
infection. Although, a statistically significant relationship
could not be established in this study between white
blood cell count and frequency of pain. Undoubtedly,
leucocytosis is associated with poor prognosis, [10-17]
while reducing neutrophil count is associated with good
prognosis. The benefit of hydroxyurea therapy in sickle
cell anaemia follows a fall in neutrophil count, even in
patients who have no increase in haemoglobin F 
Secondly, and more importantly, leucocytosis in sickle
cell disease patients may due to auto splenectomy
resulting from recurrent splenic vessels occlusion, which make
patients more vulnerable to overwhelming infections
particularly, encapsulated organisms like Streptococcus
pneumonia and Haemophilus influenzae.
Despite the controls recruitment being un-intentionally
skewed towards female gender who constituted 70% of
the volunteers, the overall mean of haemoglobin
concentration and packed cell volume of controls 13.83 ± 1.32 g/
dl and 43.07 ± 3.95% respectively almost doubles that of
cases 7.93 ± 1.47 g/dl and 24.44 ± 4.68% respectively. The
rate of chronic haemolysis associated with sickle cell
anaemia patients could account for these lower values. There
is also a blunted response to erythropoietin secretion in
sickle cell anaemia; the rate of increase is not proportional
to the degree of anaemia . This may be due to right—
shifted haemoglobin dissociation curve seen in sickle cell
disease . Similarly lower values were obtained by
Omoti in Benin city, Nigeria  amongst homozygous
sickle cell disease patients in steady state.
The mean cell volume, mean cell haemoglobin, and
mean cell haemoglobin concentration are all reduced in
anaemia of chronic disease. Expectedly, the mean
haemoglobin concentration, packed cell volume, mean
cell volume, and mean cell haemoglobin of the controls
were higher than cases and males higher than females in
both cases and controls. The effects of anaemia of
chronic disease, infections and haemolysis could account
for lower values seen in cases compared to controls.
Also, confounding factors like cigarette smoking and
alcohol intake were ruled out in the cases because
insignificant number of them gave history of cigarette
smoking and alcohol intake. Increased erythropoesis due to
androgens in males, and iron loss or blood loss in
females during menstruation may be responsible for
higher levels of the red cell indices in males. Reference
ranges for erythropoietin are however, not different
between the sex.  A negative feedback effect on
erythropoietin production in males resulting in lower
erythropoietin levels would have been expected because
of the androgen effect. This indicates that females have
better tissue oxygenation for a given haemoglobin level
and more efficient tissue red cell delivery.
The overall mean platelet counts for the cases
were 412.71 ± 145.09*103/μl and for the controls
222.82 ± 57.62*103/μl which is almost 100% higher in
cases compared to controls. A negative feedback effect
on erythropoietin production in subjects as a result of
the anaemia could be responsible for the
thrombocytosis. Erythropoietin has a structural homology with
thrombopoetin, although the latter is considerably larger
than the former but roughly half of thrombopoetin has
identity with or similarity to erythropoietin at the
Nterminal region.  It is therefore, well recognized that
thrombocytosis is associated with anaemia of chronic
disease and several types of anaemia.
Reduced or absent splenic sequestration of platelets as
a result of hyposplenism in sickle cell disease also
contribute significantly to higher mean platelet counts in
sickle cell disease compared with controls.  This
study provides haematologic reference ranges for
homozygous sickle cell disease patients compared with normal
controls in Lagos, Nigeria. It is our hope, physicians
involved in managing sickle cell anaemia patients would
become more informed and make use of the findings in
this study in their practice.
Homozygous sickle cell disease patients have lower
values of Haemoglobin concentration, packed cell
volume, red cell indices, but higher values of white cell
The authors declare that they have no competing interests.
A.A.A Conceptualized and designed the study. Did the data entry and
analysis. D.A.O Drafted the manuscript and revised it critically for important
intellectual content. A.A Made substantial contributions to conception and
design of the manuscript and revised before final submission. O.O Was
involved in the drafting of the manuscript and revised it critically for
important intellectual content. A.O - Carried out the full blood count analysis.
Dr. A.P - Revised the manuscript critically for important intellectual content
and gave final approval of the version to be published. All authors read and
approved the final manuscript.
Limitations of the study
1. All sickle cell cases were diagnosed by only haemoglobin electrophoresis
using alkaline buffer. There is no facility to confirm the diagnosis in the
centre, some of them might have thalassaemia trait e.g. SBthal which could
impact on RBC count, MCV, and haemoglobin concentration.
2. Female patients or controls were not screened for iron deficiency. Presence of iron deficiency in either patients or controls could impact on observed results.
3. Lower haemoglobin in the females may be due to menstrual blood loss.
4. Patients were also not screened for hepatitis B and C infections, known
complications of blood transfusion which could induce cytopenias in them.
5. Reliability on background information provided by patients.
6. Lack of case–control matching.
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