Influence of crisis on haemoglobin F level in adult Nigerian sickle cell anaemia patients


Olatunji, P.O.; Falusi, A.G.; Esien, E.M.

Central African Journal of Medicine 38(6): 242-246

1992


Forty-six Nigerian adult sickle cell anaemia patients were investigated, each in sickle cell crisis and steady state. Forty-three patients had vaso-occlusive crisis while three had haemolytic episodes. Investigations included Packed Cell Volume (PVC), Reticulocyte count and Haemoglobin F estimation. PCV was carried out by the microhaematocrit method while the reticulocytes were counted manually. Haemoglobin F was estimated by the Alkali Denaturation Technique. There was significant anaemia (p < 0.05) and reticulocytosis (p < 0.0001) during the period of crisis compared to the steady state. There was no significant difference (p > 0.05) between HbF level in crisis and that in the steady state. In other words the previously documented increase in HbF during reticulocyte response did not take place in our model. Maybe a 'critical' level of reticulocytosis was not attained. It was also shown that vaso-occlusive crisis did not induce an increase in HbF level suggesting that HbF might be genetically determined at a constant low level throughout life in each of our patients.

CENTRAL
AFRICAN
JOURNAL
OF
MEDICINE
Vol.
38,
No.
6,
June
1992
REFERENCES
1.
Mahmud-Durrani
A,
Bhattacharya
S
K,
Desai
M
H.
Thyroid
diseases
at
the
Central
Hospital,
Kitwe:
Review
of
212
eases.
Med
J
Zambia
1972;
May
issue:
78-87.
2.
Elcm
B,
Patil
P
S.
Pattern
of
urological
malignancy
in
Zambia,
a
hospital
based
histopathological
study.
Br
J
of
Urology
1991;
67:
37-39.
3.
Doniach
I.
The
thyroid:
Epedemiology
of
thyroid
cancer
J
R
Col
Surg
Edinb
1969
14;
5:
261-262.
4.
Singarayar
J,
Umerah
B,
Sinha
P.
Thyrotoxicosis
in
the
Zambians.
Med
J
Zambia
1973
13;
3:
50-52.
5.
Bayley
A
C.
Surgical
pathology
of
HIV
infection.
I
Psson
from
Africa.
Br
J
Surg
1990;
77:
863-868.
6.
Robbins
S.L.
Kumar
V.
The
endocrine
system
chapter
20.
In:
Dean
Manke,
Basic
Pathology
4th
ed.
Philadelphia:
W
B
Saunders,
1987;
671-703.
Elliot
A
M,
Luo
N,
Tembo
G,
et
al.
Impact
of
HIV
on
tuberculosis
in
Zambia:
A
cross
sectional
study
Br
Med
J
1990;
301:
412-415.
Influence
of
crisis
on
haemoglobin
F
level
in
adult
Nigerian
sickle
cell
anaemia
patients
PO
OLATUNIL
I
AG
FALUSI
2
,
EM
ESIEN
3
SUMMARY
Forty-six
Nigerian
adult
sickle
cell
anaemia
patients
were
investigated,
each
in
sickle
cell
crisis
and
steady
state.
Forty-three
patients
had
vaso-occlusive
crisis
while
three
had
haemolytic
episodes.
Investigations
included
Packed
Cell
Volume
(PVC),
Reticulocyte
count
and
Haemoglobin
F
estimation.
PCV
was
carried
out
by
the
microhaematocrit
method
while
the
reticulocytes
were
counted
manually.
Haemoglobin
F
was
estimated
by
the
Alkali
Denaturation
Technique.
There
was
significant
anaemia
(p<0.05)
and
reticulocytosis
(p<0.0001)
during
the
period
of
crisis
compared
to
the
steady
state.
There
was
no
significant
difference
(p>0.05)
between
HbF
level
in
crisis
and
that
in
the
steady
state.
In
other
words
the
previously
documented
increase
in
HbF
during
reticulocyte
response
did
not
take
place
in
our
model.
Maybe
a
`critical'
level
of
reticulocytosis
was
not
attained.
It
was
also
shown
that
vaso-occlusive
crisis
did
not
induce
an
increase
in
HbF
level
suggesting
that
HbF
might
be
genetically
determined
at
a
constant
low
level
throughout
life
in
each
of
our
patients.
'Department
of
Haematology
University
of
Bonn
2
Postgraduate
Institute
of
Medical
Research
and
Training
College
of
Medicine
University
of
Ibadan
3
Dir
.
ector
National
Institute
for
Medical
Research
Lagos
Correspondence
to:
Dr
PO
Olatunji
Department
of
Haematology
Faculty
of
Health
Sciences
University
of
florin
PMB
1515
!Lorin
Nigeria
242
CENTRAL
AFRICAN
JOURNAL
OF
MEDICINE
Vol.
38,
No.
6,
June
1992
INTRODUCTION
Sickle
cell
anaemia
patients
have
been
known
to
have
a
significantly
higher
level
of
Haemoglobin
F
(HbF)
than
normal
individuals.'
Patients
who
have
in
addition
Hereditary
persistence
of
Foetal
Haemoglobin
(HPFH)
or
SR
Thalassaemia,
are
associated
with
HbF
levels
ranging
from
17-35pc
and
milder
clinical
courses.
2
'
3
These
genetic
traits
have
not
been
observed
in
Nigeria
where
the
mean
HbF
level measured
in
steady
state
was
found
to
be
low.
4
Apart
from
the
above
genetic
influence
on
the
level
of
HbF,
patients
who
have
anemia
from
other
causes
such
as
Pernicious
Anaemia,
Leukamia,
Hereditary
Spherocytosis
and
those
recovering
from
bone
marrow
transplantation
were
observed
to
have
high
HbF
levels.
5
In
these
patients,
it
is
believed
that
the
anaemia
created
a
form
of
erythropoietic
stress
which
caused
a
physiological
increase
in
the
level
of
erythropoietin.
6
This,
it
was
suggested,
was
then
followed
by
the
production
and
release,
by
the
bone
marrow,
of
reticulocytes
containing
higher
levels
of
HbF.
7
Sickle
cell
anaemia
patients,
who
are
chronically
anaemic,
share
the
above
mechanism
as
part
of
the
explanation
for
the
observed
higher
level
of
HbF.
In
addition,
it
had
been
suggested
that
during
sickle
cell
vaso-occlusive
crisis,
cells
with
low
HbF
content
were
preferentially
irreversibly
sickled
and
sequestered
thus
leaving
behind
cells
with
higher
HbF
content!
It
seems
logical
therefore
to
expect
that
during
sickle
cell
crisis
associated
with
sickling
and
significant
reduction
in
haematocrit,
HbF
level
should
increase
further.
Previous
studies
aimed
at
determining
whether
or
not
HbF
levels
fluctuated
significantly
from
crisis
to
steady
state
periods
had
not
produced
consistent
results.
While
one
study
reported
a
significant
fall
in
HbF
level
crisis,
8
another
study
observed
no
significant
change
in
HbF
levels.
9
We
therefore
proceeded
by
studying
our
own
patients
in
Nigeria
both
in
crisis
and
steady
state
with
the
aim
of
testing
the
above
hypothesis
and,
or,
reproducing
any
of
the
results
in
the
two
cited
studies.
The
haematocrits,
reticulocyte
counts
and
HbF
levels
were
determined
in
the
two
periods
and
comparisons
made.
The
results
obtained
enabled
us
to
determine
the
validity
of
the
various
mechanisms
described
with
respect
to
our
patients.
The
pc
HbF
was
also
correlated
with
the
reticulocyte
counts
in
both
crisis
and
steady
state
periods.
MATERIALS
AND
METHODS
Patients:
Patients
included
sickle
cell
anaemia
patients
who
were
being
seen
regularly
at
the
Haematology
Department
of
the
University
College
Hospital,
Ibadan.
The
patients
had
been
diagnosed
by
demonstration
of
positive
sickling
phenomenon
and
Hb
Electrophoresis.
Patients
were
investigated
during
the
crisis
period
and
at
least
four
weeks
later
when
in
the
steady
state.
Patients
were
categorised
as
having
vaso-occlusive
crisis
if
the
presenting
feature
was
limited
to
bone
pains,
or
haemolytic
when
there
was
severe
anaemia
plus
unconjugated
hyper-
bilirubinaemia
of
above
10mg/litre.
Two
and
a
half
millilitres
of
blood
was
collected
from
each
patient
via
the
antecubital
vein
after
obtaining
informed
consent.
The
samples
were
collected
in
bottles
containing
Ethylene
Diamine
Tetraacetic
Acid
(TDTA)
An
aliquot
was
used
to
determine
the
packed
cell
volume
(PCV)
and
reticulocyte
count
while
the
remainder
was
used
to
prepare
haemolysate
by
the
method
of
Wood.
Methods:
Haemolysate
was
prepared
from
sequestrene
sample.
The
red
blood
cells
were
washed
three
times
in
normal
saline
and
the
supernatant
was
completely
removed
after
the
third
wash.
Two
volumes
of
distilled
water
was
added
to
one
volume
of
the
washed,
packed
cells
and
a
few
drops
of
carbon
tetrachloride
added.
The
mixture
was
shaken
vigorously
for
about
30
seconds
and
centrifuged
at
3000
r.p.m.
after
which
the
haemolysate
was
transferred
into
another
bottle.
HbF
level
was
determined
from
the
haemolysate
by
the
Alkali
Denaturation
technique
described
by
Becke
tt
and
modified
by
Penbery
a
at
i2
The
mean
PCV,
Reticulocyte
count
and
HbF
level
during
crisis
and
steady
state
periods
were
compared
for
significance
using
the
students
't'
test.
The
Correlation
Co-efficient
between
reticulocyte
count
and
pc
HbF
was
also
determined during
crisis
and
steady
state
respectively.
243
CENTRAL
AFRICAN
JOURNAL
OF
MEDICINE
Vol.
38,
No.
6,
June
1992
SUMMARY
Forty-six
out
of
58
patients
were
evaluated.
These
were
made
up
of
19
males
and
27
females
aged
14-33
years.
Of
the
46
patients,
three
had
haemolytic
crisis
while
43
had
vaso-occlusive
crisis.
Table
I:
Mean
PCV,
Reticulocyte
Count
and
pc
HbF
in
crisis
and
steady
state.
Crisis
Steady
state
t
value
Mean
PCV
(pc)
23.13±5.26
25.49±4.29
2.36
0.05
Mean
Reticulocyte
10.27±5.5
6.29±3.16
4.23
0.0001
Count
Mean
pc
HbF
3.67±2.0
3.48±1.96
0.46
0.05
Table
I
shows
that
the
mean
PCV
was
23.13±5.26pc
and
25.49±4.29pc
during
the
crises
period
and
steady
state
respectively.
The
mean
reticulocyte
count
was
10.27±5.55pc
during
the
crisis
period
and
6.29±3.16pc
during
the
steady
period.
The
mean
HbF
levels
were
3.67
-
±2.0pc
and
3.48±96pc
in
crisis
and
steady
state
respectively.
The
mean
PCV
was
significantly
reduced
(t
value
=
2.6,
p<0.05)
and
the
mean
reticulocyte
count
significantly
increased
(t
value
=
4.23,
p<0.0001)
during
the
period
of
crisis.
The
pc
HbF
did
not
show
any
significant
difference
(t
value
=
0.46,
p>0.05.
Table
II:
Effect
of
type
of
crisis
on
fluctuation
of
mean
HbF
level.
n
pc
HbF
Crisis
Steady
state
t
value
P
Vaso-occlusive
43
3.48±1.9
3.46±1.8
0.05
0.5
Haemolytic
3
6.48±1.37
4.16±1.56
1.94
0.05
Table
II
shows
the
mean
pc
HbF
during
the
periods
of
crisis
and
steady
state
with
regard
to
the
of
Crisis.
For
the
patients
with
vaso-occlusive
crisis,
the
Mean
pc
HbF
were
3.67
-
±2.0pc
and
3.48±1.96pc
during
the
periods of
crisis
and
steady
state
respectively
(t
value
=
0.05,
p>0.05).
In
the
case
of
the
only
three
patients
with
haemolytic
crisis,
the
mean
pc
HbFs
were
6.48±1.32pc
and
4.16±1.56pc
during
the
period
of
crisis
and
steady
state
respectively.
Figures
I
and
II
show
scatter
diagrams
of
reticulocyte
counts
against
pc
HbF
in
both
crisis
and
steady
state
periods.
The
correlation
co-efficients
were
0.0913
(p
=
0.546)
and
0.1014
(p
=
0.503)
respectively.
These
show
no
statistical
significance.
DISCUSSION
There
is
a
paucity
of
studies
on
I-IbF
level
in
Nigeria
sickle
cell
anaemia
patients.
The
major
study
so
far
is
that
of
Falusi
and
Esan
4
in
which
the
mean
pc
HbF
in
patients
age
two
to
30
years
was
found
to
be
5.9±3.8pc.
The
mean
pc
I-IbF
obtained
in
the
present
study
is
lower
than
the
above
value.
The
difference
is
probably
due
to
the
fact
that
only
adult
patients
were
studied,
and,
our
patients
having
been
studied
in
crisis,
may
be
made
up
of
those
with
lower
levels
of
HbF.
The
observation
in
this
study
shows
that
sickle
cell
crisis,
either
vaso-occlusive
or
haemolytic,
was
associated
with,
significant
reduction
in
haematcrit
(p>0.05)
as
well
as
significant
reticulocytosis
(p<0.0001).
One
would
expect
that,
in
line
with
suggested
mechanisms,
there
should
have
been
associated
increases
in
the
HbF
level.
However,
our
study
did
not
demonstrate
any
significant
difference
between
HbF
levels
in
crisis
and
steady
state
periods
in
patients
with
vaso-occlusive
crisis.
Our
result
is
at
variance
with
the
findings
of
Morrison
et
al
who
observed
a
fall
in
the
level
of
HbF
during
the
period
of
crisis.
The
result
of
Morrison
is
difficult
to
explain
considering
the
available
theories
of
the
effect
of
reticulocytosis
and
preferential
sickling
of
red
cells
with
low
HbF
level
during
sickle
cell
crisis.One
would
have
expected
an
increase
in
HbF
level
rather
than
a
fall
if
them
was
going
to
be
any
fluctuation
during
the
period
of
crisis.
Our
result
is
in
agreement
with,
and
a
confirmation
of
the
finding
of
Steinberg
and
Adams,
who
could
not
observe
any
fluctuation.
Our
observation
however,
raised
doubt
as
to
the
validity
of
the
effect
of
reticulocytosis
and
preferential
sickling
of
red
cells
with
relatively
low
HbF
levels.
It
also
implied
that
an
increase
in
HbF
may
not
be
involved
in
any
homeostatic
mechanism
to
reverse
the
crisis
situation.
With
respect
to
the
only
three
patients
seen
in
haemolytic
crisis,
the
mean
pc
HbF
in
crisis
and
steady
state
did
not
show
any
significant
difference
244
a
a
a
a
a
C
a
HbF
.B7
0
Reticulocyte
Count
Slope
:
29
0
0a9
9
E-0
9
:
q.
4
0
7v.0
1
'2R
Cnrrin
cneff
:
0.09
1
3
CENTRAL
AFRICAN
JOURNAL
OF
MEDICINE
Vol.
38.
No.
6,
Lune
1992
Figure
I:
Scatter
diagram
showing
correlation
between
pc
HbF
and
Reticulocyte
count
in
crisis.
Figure
II:
Scatter
diagram
showing
correlation
between
pc
HbF
and
Reticulocyte
count
in
steady
state.
R
.17
a
%
HbF
a
c
a
a
a
a
2
a
0
Reticulocyte
count
1.1
5.291724E-02
Intercept
:
3.
1
00.11
Cnrrin
r..aFf
-
0
.
1014
245
4
ENTRAL
AFRICAN
URNAL
OF
MEDICINE
VoL
38.
No.
6,
June
1992
ven
though
the
number
is
too
small
for
valid
6.
Desimore
J,
Biel
M,
Heller
P.
Maintenance
of
nclusion.
It
will
be
necessary
to
study
HbF
in
Foetal
Haemoglobin
(HbF)
Elevation
in
the
pa
tients
with
haemolytic
anaemia
from
causes
other
baboon
by
prolonged
Erythropoietic
stress.
than
sickle
cell
anaemia
both
during
acute
haemolytic
Blood
1982;
60:
519-523.
ph
ase
and
stable
state.
7.
Bertles
JP,
Milner
PFA.
Irreversibly
sickled
The
only
other
explanation
for
our
observation
in
erythrocytes:
A
consequence
of
heteroge-
patients
with
vaso-occluiive
crisis
is
that
'critical'
neous
distribution
of
Haemoglobin
F
types
in
ticulocytosis
necessary
for
increase
in
HbF
level
ight
not
have
been
attained.
Our
finding
of
'
l
ati
Sickle
Cell
Anaemia.
J
Clinic
Invest
1968;
47:
1731-1741.
significant
correlation
between
reticulcyte
couni
d
HbF
further
confirms
the
inapplicability
of
the
8.
Morrison
JC,
Whyhrew
WD,
Bucovaz
ET,
Wiser
WH.
Fluctuation
of
Foetal
Haemo-
perimental
models
cited
to
our
clinical
model.
globin.
Am
J
Obstet
Gynaecol
1976;125:
Overall,
our
findings
suggest
that
in
adult
sickle
1085-1088.
cell
anaemia
patients,
HbF
level
is
not
affected
by
9.
Steinberg
MH,
Adams
JG.
Haemoglobin
F
isodic
occurrences
of
crisis
and
therefore
can
not
to
predict
either
the
imminence
or
presence
levels
do
not
change
during
painful
crisis
of
Sickle
Cell
Anaemia.
Am
J
Obstet
Gynaecol
f
used
crisis
or
the
response
of
same
to
instituted
1977;
129:
712-713.
anagement.
10.
Wood
WG.
Haemoglobin
Analysis.
In:
Weatherall
DJ.
The
Thalassaemias
1st
ed.
ACKNOWLEDGEMENTS
Churchill-Livingstone,
1983;
31-53.
e
are
grateful
to
Mr
Ipadeola,
Assistant
Chief
edical
Laboratory
Technologist
in
the
Department
11.
Betke
K,
Marti
HR,
Schhcht
K.
Estimation
of
small
percentage
of
Foetal
Haemoglobin.
Nature
1959;
184:
1877-1878.
Of
Haematology
for
his
assistance.
Ms
JA
Otitoju
kindly
typed
the
script.
REFERENCES
12.
Pembery
ME,
McWade
P,
Weatherall
DJ.
Reliable
routine
estimation
of
small
amounts
of
Foetal
Haemoglobin
by
Alkali
Denaturation.
J
Clin
Pathol
1972;
25:
1.
Huisman
THJ.
Normal
and
Abnormal
738-740.
Haemoglobins.
Advance
in
Clinical
13.
Heller
P,
De
Simone
J.
Five-Azacitidine
and
Chemistry
1963;
6:231
7
361.
Foetal
Haemoglobin.
Am
J
Haematol
1983;
Pembery,
ME,
Wood
WG,
Weatherall
DJ
et
al.
17:
439-447.
Foetal
Haemoglobin
production
and
Sickle
14.
Charache
S,
Dover
GJ,
Moyer
MA,
Moore
genes
in
the
Oasis
of
Eastern
Saudi
Arabi.
Brit
JW
Hydroxyrea
Induced
Augmentation
of
J
Haematol
1978;
40:
415-429.
Foetal
Haemoglobin
production
in
patients
Wood
WG,
Pembery
ME,
Serjeant
GR
et
al.
with
Sickle
Cell
Anaemia.
Blood
1987;
69:
HbF
synthesis
in
Sickle
Cell
Anaemia.
A
comparison
of
Saudi
Arab
cases
with
those
of
109-116.
African
Origin.
Brit
.
J
Haematol
1980;
45:
431-445.
Falusi
AG,
Esan
GJF.
Foetal
tiaemoglobin
in
sickle
cell
anaemia
in
Nigerians.
AfrJ
Med
Sci
1989;
18;
145-149..
S.
Singer
K,
Chemoff
AI,
Singer
L.
Studies
on
Abnormal
Haemoglobins:
The
Demon-
stration
in
SCA
and
other
Haematological
Disorders
by
means
of
Alkali
Denaturation.
Blood
1951;
6:
413-428.
246