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Comparative Erythrocytes Osmotic Fragility Test and some Haematological

Parameters in HbAA and HbSS subjects

Article  in  Journal of Medical Laboratory Science · September 2007

DOI: 10.4314/jmls.v14i1.35309

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 Journal of Medical Laboratory Science 2005 Vol. 14 No.1'

Comparative Erythrocytes Osmotic Fragility Test and some Haematological

Parameters in HbAA and HbSS subjects

S.O. ITA* AND B.A. AKPOGOMEH

"Department of Physiology, College of Health Sciences, University of Uyo, Akwa Ibom State,
Nigeria and 2 Department of Physiology, College of Medical Sciences, University of Calabar,
Calabar.
E-mail.·uloro2003@yahoo.com

ABSTRACT
Erythrocyte osmotic fragility and haematological parameters of subjects with HbAS (sickle
cell trait) and HbSS (sickle cell anaemia) were determined and compared with subjects with HbAA
(normal adult haemoglobin), which acted as control. They were divided into three groups of 40
subjects for HbAA, 35 subjects for HbAS and 30 for HbSS. HbAA erythrocytes showed a higher
degree of susceptibility to lysis than both HbAS and HbSS erythrocytes in buffered hypotonic
sodium chloride solution. There were variations in the profiles of osmotic fragility curves for
HbAA, although there was no significant difference in their MCF values. It was observed that
HpAA erythrocytes completely haemolysed under 12.24 ± 0.25 minutes (n = 40) at 0.60gldl
sodium chloride solution. This value was significantly lower than that of HbAS which was 21.02 ±
0.32 minutes and HbAS in turn which was significantly lower than HbSS as well (p <: 0,0001).
Erythrocytes with HbSS type showed corpuscular haemaglobin concentration that was significantly
lower than HbAA and HbAS (p < 0.0001); mean values of HbAA and HbAS did not show any
significant difference. In contrast, HbAA erythrocytes showed mean corpuscular volume (MCV)
value that was significantly lower than those of HbAS and HbSS (p < 0.001). Although the MCV
value of HbAS erythrocytes was lower than that of HbSS, the difference was not statistically
significant. The study showed that the presence of the sickle haemaglobin (HbS) whose structure
conferred on the cell a larger volume as indicated by the highest mean MCV value coupled with its
reduced corpuscular haemoglobin content may account for the left-ward shift of HbAS and HbSS
curves. Again, erythrocytes with HbS will normally take a longer time to reach and exceed their
critical haemolytic volume than those with HbA type, with a resultant effect that HbSS erythrocytes
of healthy subjects are more resistant to lysis than HbAS and HbAA in buffered hypotonic sodium
chloride solution.

Keywords: Erythrocytes, osmotic fragility, haematological parameters.

INTRODUCTION
The normal mammalian red blood cell
membrane is permeable to fluid and has the
ability to assume critical volume when
suspended in a saline medium. As fluid diffuses
into the cell, it swells to assume these varied
volumes until it reaches its "critical haemolytic
volume" after which the cell lyses or bursts to
release its content into the surrounding fluid.
The osmotic fragility of human mammalian
erythrocytes suspended in a hypotonic saline
mediumhas been reported to be indicative of
their viability. Alterations in osmotic fragility
of human erythrocytes is used as an important
diagnostic technique in disease conditions as in
hereditary spherocytosis, betathalasaemia,
sickle cell anaemia and autoimmune haemolytic
anaemia (1-5).
This study was designed to make a
comparative study of the integrity of HbA and
HbS cells in various concentrations of buffered
hypotonic sodium chloride solution and to
possibly find out how much time HbAA,
HbAS
and HbSS erythroctye cell will require to
absorb
fluid that exceed their critical hymolytic
volume
before they haemolyse.
MATERIALS AND METHODS,
In this study, venous blood from 105
apparently healthy subjects (40 AA, 35 AS and
30 SS) between the ages of nine and twenty
years of both sexes were subjected to screening
using cellulose acetate electrophoresis
technique. The same samples were thereafter
subjected to osmotic
Journal of Medical Laboratory Science 2005 Vol. 14 No. 1
fragility test and determination of haemoglobin
content, packed cell volume and red blood cell
count. The blood samples for the test groups
were collected into EDTA sample bottles from
subjects attending sickle cell clinics for
counseling and routine medical check-up at the
University of Calabar Teaching Hospital
(UCTH), Medical Center of the University of
Calabar and General Hospital, Calabar. The
control groups were apparently healthy subjects
in Calabar metropolis. From each subject, 5ml
of venous blood was withdrawn from the
antecubital vein. The samples were subjected to
electrophoresis to determine their haemoglobin
status.
Osmotic fragility test
Osmotic fragility test of the erythrocytes was
determined according to the method described
by Dacie and Lewis (6). Stock 10% buffered
sodium, chloride (NaCl) solution (pH 7.4) was
prepared by dissolving NaCI, 18g;
NaH2P04.2HzO, 0.486g; Na2HP04, 2.731g
and made up to 1000 ml with distilled water. A
1 % saline solution was prepared from the
stock solution by diluting 1 in 10 with distilled
water. From the 1 % saline solution, serial
dilutions were made to obtain the various test
concentrations of
0.10,0.20,0.30,0.35,0.40,0.45,0.50,0.60, 0.65,
0.70,0.80 and 0.90gldl, using this formula:
R soln X RV
S soln
Where R soln is the required saline
concentrtion, S soln is the stock saline
concentration RV is the required volume
(lOOml) These were then made up to l00ml
with distilled water. A set of test tubes labeled
1 - 13 were set up on a rack. To each of tubes 1
- 12, 5ml of the test concentrations was added
accordingly, while 5ml of 0.9g/ml buffered
sodium chloride solution was added to the 13th
tube (serving as blank). Of each well mixed
blood specimen 0.05 ml was added accordingly
to the remaining 12 tubes, after which the tubes
were inverted to mix and allowed to stand at
room temperature (25°C) for 30 minutes. At 5
minutes intervals, 0.5ml of blood dilution was
drawn from test tubes containing HbAA,
HbAS, HbSS specimens to find out which
takes a shorter time to completely haemolyse.
At these intervals, blood dilutions were
withdrawn with pipette and placed on a slide to
check microscopically for the presence of red
blood cells that were yet to haemolyse. At the
end of 30 minutes, test tubes were centrifuged
for 5 minutes at 1200g. The supernatant from
each test tuhes was carefully decanted into
freshly labeled lubes for estimation of
percentage lysis occurring in each tube using a
spectrophotometer at a wave length of 540nm.
The haemoglobin concentration, packed cell
(PCV) volume and red blood count were
·determined according to the methods of Dacie
and Lewis (6).
Statistical Analysis
Analysis of variance (ANOVA) was used in all
analyses involving comparison of the three
groups for significant difference. Students t-
test for non-paired samples was adopted for all
analyses involving comparison of the control
group (HbAA) and either of the test groups.
RESULTS
Figure 1 shows osmotic fragility or mean
percentage haemolysis of HbAA, HbAS and
HbSS erythrocytes against buffered sodium
chloride concentrations from O.lg/dt'- 0.9g1dl.
The HbAA erythrocytes were more fragile
than HbSS erythrocytes. Similarly, HbAS
erythrocytes were more fragile than HbSS
erythrocytes. The median corpuscular fragility
of the three groups varied but showed no
significant differences (Tables 1,2 and 3). The
mean haemoglobin concentration
 Journal of Medical Laboratory Science 2005 Vol. 14 No. 1 3

Table I Mean Haematological values and median corpuscular

packed cell volume and red blood cell counts of fragility of subjects with HbAA and HbAS
both HbAA and HbAS erythrocytes showed no
significant differences, except mean corpuscular Parameters HbAA HbAS P -value
(n=40) (n=35)
volume that was significantly lower than that of
Hb concentration (gldl) 12.25±0.23 11.54±0.30 NS
HbAS erythrocytes (89.10 ± 1.43), (p < 0.001). PCV(%) 37.15±0.71 36.00±0.79 NS
Table 1. The fragility curve of HbAS shifted to RBC (Millions/mm ') 4. 19±0. 10 3,91±0.12 NS
Mean Corpuscular volume (IL') 89,1O±0.98 93.12±0.98 <0.001
the left in accordance with its reduced Hb Median Corpuscular Fragility 46.63±6.43 44.35±6,92 NS
concentration (Figure 1).
The Hb concentration, PCV and red cell
counts ofHbAA erythrocytes were observed to
be significantly higher than those of HbSS
erythrocytes (P< 0.0001), except the MCV that
Table 2 Median Haematological values and median
was significantly lower than that of HbSS corpuscular fragility of subjects with HbAA and HbSS
erythrocytes (89.10 ± 0.98 vs 95.54 ± 2.45/13)
(P<O.OOl) Table 2. The fragility curve of HbSS Parameters HbAA HbSS P-value
(n=40) (n=30)
shifted farther left in accordance with its
pronounced reduction of Hb concentration Hb concentration(gldl) 12.25±0.23 7.07±1.22 <0.0001
PCV(%) 37.15±0.71 24.03±0.78 <0.0001
(Figure 1). RBC (Millions/mm-) 4.19±0.1O 2.52±0.06 <0.001
Mean Corpuscular volume (IL3) 89.1O±0.98 95.54±2.45 <0.001
. Similarly, Hb concentration PCVand red Median Corpuscular Fragility 46.63±6.43 40.01±7.37 NS
blood cell counts of HbAS . erythrocytes were.
significantly higher than those of HbSS
erythrocytes (P<O.OOO1), but their MCV shows
no significant differences (Table 3). The fragility
curve of HbSS shifted to left of HbAS curve in Table 3 Mean Haematological values and median corpuscular
accordance with its reduced concentration fragility of subjects with HbAS and HbSS

(Figure 1). Parameters HbAS HbSS P-value

Table 4 showed the duration taken for (n=35) (n=30)

complete lysis of HbAA erythrocytes to be Hb concentration (g/dl) 11.54±0.30 7.07±1.22 <0.0001

. PCV (%) 36.00±0.79 24.03±0.06 <0.0001
significantly lower than both HbAS and HbSS RBC (Millions/rom') 3.91±0.12 2.52±0.06
<0.001
(P<O.OOO 1). The duration taken for complete Mean Corpuscular volume (IL') 93.12±1.43 95.54±2.45
NS
Median Corpuscular Fragility 44.35±6.92 40.01±7.37
lysis of HbAS erythrocytes was also shown to NS

be significantly lower than HbSS erythrocytes

(p < 0.0001).

Table 4 Comparison of length of time taken for complete

100
haemolysis for HbAA, HbAS and HbSS erythrocytes
'" KEY
~HbAA
80 --G>--&- HbAS
--Hb55
Haemoglobin Time of complete P-value
70 Types haemolysis (rnin.)

*'" AA (n =40) 12.24±0.25 <0.0001

if<) AS (n = 35) 21.02±0.32

~ zo
10
AA (n,",40)
SS (n=30)
AS (n = 35)
12.24±0.25
25.27±0.20
21.02±O.32
<0.0001

<0.0001
0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
SS (n = 30) 25.27±0.20
NaCl Concentration (g/dl)
Fig. I: Osmotic fragility curves for HbAA, HbAS and HbSS
 Journal of Medical Laboratory Science 2005 Vol. 14 No.1'
DISCUSSION
Deviations from normal osmotic fragility
pattern of human erythrocytes have been
observed in haemolytic disorders and have been
used to establish disease processes which affect
primarily the physical integrity of the
erythrocytes. Thus osmotic fragility test is
adopted as an important disgnostic technique in
disease conditions. The inherent properties of
erythrocytes like the shape and membrane
structures exhibit a pronounced influence on their
fragility (7,8). In the present study involving
healthy HbAA,HbAS and HbSS subjects, we
compared the integrity of HbA and HbS cells
suspended in various concentrations of buffered
hypotonic sodium chloride solution, and the time
required for excess fluid absorption and osmotic
stress to induce lysis.
The' results showed that HbAA
erythrocytes were more fragile or exhibited less
osmotic resistance in a bufferedhypotonic sodium.
chloride solution than both HbAS and HbSS
erythrocytes. Under the same experimental
conditions and timing, the fragility curves for both
HbAS and HbSS shifted to the left of the HbAA
curve. This effect might have been due to the
reduced corpuscular haemoglobin content in both
HbAS and HbSS and is consistent With earlier
reports (3,9,10). While we noted a uniform shift of
the- three curves to the right that might have been
due to the anticoagulant (EDTA) used in this
study, the increased resistance exhibited by both
HbAS and HbSS could presumably be due to the
presence of abnorrnal HbS (9) and the MCV
values that were significantly higher than that of
HbAA.
The nett effect of higher MCV is the
larger corpuscular volumes observed in HbAS
and HbSS and hence the longer time required to
exceed the critical haemolytic volume before cell
bursting. Thus the duration for complete lysis to
occur was significantly shorter in HbAA than in
HbAS and HbSS types. Similarly, the duration
for complete cell lysis was shorter in HbAS than
HbSS, a variation that was presumably due to the
larger corpuscular volume conferred by the
presence of abnormal HbS.
For all mammalian red cells, a linear
4
relationship exists between cell' volume and
maximum possible surface area. Because the
behaviour of erythrocyte in a buffered hypotonic
saline depends on the initial volume to surface
ratio and not on absolute size of the cell, the
greater the ratio, the greater the additional
volume that can be accommodated (6). Thus, a
cell with a larger volume will ordinarily take a
longer time to imbibe fluid to exceed its critical
haemolytic volume than a cell with a smaller
volume. These results agree with
the reports of previous workers (3,11) that larger
cells are more resistant to lysis than smaller ones.
The osmotic fragililgram in a sickle cell
anaemia has been reported to always shifty to the
left, suggestive of a higher degree of osmotic
resistance for most sickle cells (12). The very
low haemoglobin content, low PCV and low
osmotic fragility (or higher osmotic resistance) .
presented by HbSS erythrocytes from apparently
healthy subjects as presented here agree with the
earlier reports (7,13), that sickle cell patients are
known to present a reduced haemoglobin
concentration, packed cell volume and
irreversibly sickle cells (ISC).
In conclusion, we report that osmotic
fragility of erythrocytes may be a direct function
of the haemoglobin type, intracellular
haemoglobin concentration, intracorpuscular
volume and several other independent variables.
The present findings have provided information
on the basic pattern of the osmotic fragility of
apparently healthy heterozygous HbAS .and
homozygous HbSS subjects with respect to
homozygous HbAA subjects. The decreased
fragility presented by HbAS and HbSS is a .
function of the mutant haemoglobin S which
confers a larger critical haemolytic volume on
both HbAS and HbSS types than HbAA type.
This is reflected on time taken for complete lysis
of each of the three haemoglobin types, with
HbSS type subjects having the capacity to stay
longer in a hypotonic sodium chloride solution
before bursting. Therefore, in a non-crisis state,
erythrocytes with HbS take a longer time to
completely
Journal of Medical Laboratory Science 2005 Vol. 14 No. 1 5

haemolyse when suspended in a buffered parts). New Engl. J. Med. 299

hypotonic sodium chloride solution than 804 - 811.
HbA.

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