LETTER TO THE EDITOR OSMOTIC FRAGILITY OF NORMAL AND … archives/1999_43_2/267-269.pdf · 2018. 8....
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Indian J Physiol Pharmacol 1999; 43 (2) : 267-269 LETTER TO THE EDITOR OSMOTIC FRAGILITY OF NORMAL AND SICKLE HAEMOGLOBIN CONTAINING RED BLOOD CELLS Sir, (Received on June 9, 1998) Abnormalities in osmotic fragility have been observed in hereditary spherocytosis, hereditary elliptocytosis, pyruvate kinase deficiency, thalassaemia and iron-deficient red cells (1). Harris et al have reported a decrease in osmotic fragility of the red cells of sickle cell anaemia patients from Africa (2). The Indian patients are genetically and clinically different from the African patients (3). Though the abnormalities in the glucose metabolism and a greater loss of K+ ion from the red blood cells of our patients during incubation have been reported (4, 5), data pertaining to the osmotic fragility of these cells are not available. Therefore, we have estimated the osmotic fragility curve of both fresh and incubated red blood cells of the homozygous sickle cell disease patients. Blood smaples from 8 adult normal healthy subjects, 11 healthy sickle cell trait cases and 10 homozygous sickle cell disease patients were collected. The patients were in steady state at the time of blood collection and no patient had received transfusion during the last three months prior to the study. The haemoglobin (mean ± SEM) for normal (AA), sickle cell trail (AS) and sickle cell patients (SS) subjects were 14 ± 0.34 g/dl, 13.2±1.4 g/dl and 10.9±1.0 g/dl respectively. The reticulocyte counts (mean±SEM) of AA, AS and SS sample were 0.64±0.26%, 1.2±0.57% and 6.9±3.5% respectively. The sickle cell disease patients had lower hamoglobin level (P<O.OOI) and higher reticulocyte count (P<O.OOl) than the normal and sickle cell trait cases. Immediately after the blood collection, the fresh red cell osmotic fragility test was performed in all the test samples. 1 ml of whole blood was incubated in sterile tubes at 37°C in a water bath for 24 hours. After incubation the red cell osmotic fragility test was again done for all three groups of samples. Osmotic fragility test was performed (1) using haparinised venous blood. The O.D. was measured in Elico Digital UV spectrophotometer (Model CL - 54 D) at a wave length of 540 nm. Percentage of lysis of red blood cells was then calculated. Fragility curves were drawn taking percentage (%) of lysis against the (%) of NaCl solution. The data were analysed by Student's It' test. The concentration of NaCl solution in which 50% lysis occured or the median corpuscular fragility (MCF) point for fresh AA, AS, SS red cells were found to be at 0.41 %, 0.38% and 0.33% respectively. After
Transcript of LETTER TO THE EDITOR OSMOTIC FRAGILITY OF NORMAL AND … archives/1999_43_2/267-269.pdf · 2018. 8....
Indian J Physiol Pharmacol 1999; 43 (2) : 267-269
LETTER TO THE EDITOR
OSMOTIC FRAGILITY OF NORMAL AND SICKLE HAEMOGLOBINCONTAINING RED BLOOD CELLS
Sir,
(Received on June 9, 1998)
Abnormalities in osmotic fragility havebeen observed in hereditary spherocytosis,hereditary elliptocytosis, pyruvate kinasedeficiency, thalassaemia and iron-deficientred cells (1). Harris et al have reported adecrease in osmotic fragility of the red cellsof sickle cell anaemia patients from Africa(2). The Indian patients are genetically andclinically different from the African patients(3). Though the abnormalities in the glucosemetabolism and a greater loss of K+ ion fromthe red blood cells of our patients duringincubation have been reported (4, 5), datapertaining to the osmotic fragility of thesecells are not available. Therefore, we haveestimated the osmotic fragility curve of bothfresh and incubated red blood cells of thehomozygous sickle cell disease patients.
Blood smaples from 8 adult normalhealthy subjects, 11 healthy sickle cell traitcases and 10 homozygous sickle cell diseasepatients were collected. The patients werein steady state at the time of blood collectionand no patient had received transfusionduring the last three months prior to thestudy. The haemoglobin (mean ± SEM) fornormal (AA), sickle cell trail (AS) andsickle cell patients (SS) subjectswere 14 ± 0.34 g/dl, 13.2±1.4 g/dland 10.9±1.0 g/dl respectively. The
reticulocyte counts (mean±SEM) of AA, ASand SS sample were 0.64±0.26%, 1.2±0.57%and 6.9±3.5% respectively. The sickle celldisease patients had lower hamoglobin level(P<O.OOI) and higher reticulocyte count(P<O.OOl) than the normal and sickle celltrait cases. Immediately after the bloodcollection, the fresh red cell osmotic fragilitytest was performed in all the test samples.1 ml of whole blood was incubated in steriletubes at 37°C in a water bath for 24 hours.After incubation the red cell osmoticfragility test was again done for all threegroups of samples.
Osmotic fragility test was performed (1)
using haparinised venous blood. The O.D.was measured in Elico Digital UVspectrophotometer (Model CL - 54 D) at awave length of 540 nm. Percentage of lysisof red blood cells was then calculated.Fragility curves were drawn takingpercentage (%) of lysis against the (%) ofNaCl solution. The data were analysed byStudent's It' test.
The concentration of NaCl solution inwhich 50% lysis occured or the mediancorpuscular fragility (MCF) point for freshAA, AS, SS red cells were found to be at0.41 %, 0.38% and 0.33% respectively. After
268 Letter to the Editor Indian J Physiol Pharmacal 1999; 43(2)
NaCI SOLUTION (")
Normal (AA)
~ Sickle cell trait ( 11$)
--a- Sickle cell <lisea.e($S)
100
90
80
70
LGO
ySI 50S
:7- 40
30
20
10
o L-.L-:....-L.--l....:...L~~~~-lOl--~H0.1 0.2 0.3 0.1 0.5 0.6 0.7 0.8 O.D
Noel SOLUTION (%)
Normal (AA)
-+- Sickle cell lrail (ilS)
~ Sicklc cell di.ease(S")
70 -
100 '-
1
:: r
80T.ySI 50 - - - - - - - -'
S I : I
:I 101- , I', I
I ' I
:: f :10 rOL----l_----1....L---l.-J.~....::::E~~~_lil_lf;--L__Ec'_4!
0.1 O.~ 0.3 0.1 0.5 0.6 0.7 0.8 0.9
Fig. 1 Fr ';;h blood osmotie frarrility curv ,values arem 'an :t: • EM, ·P<O.05 Vs uoth AA & AS,• P<O.OOI Vs both A & AS.
Fig. ~: Post incubation osmotie fragility curve, valuesare mean ± SEM, ·P<0.05 Vs AA and *P<O.OOlVs AS.
2 ~ hours f incubatiull the MCl" valu()~ for, A and AS red cells were 0.4:1 1,1, .45'1":1nd for SS it was at 0.?6% NaCI :::0 utilln(Fi}.T. 1 & 2).
of AS red cells were not different than the.L A reel cells both in fresh and incubatedconditions.
It was observed that the osmotic fru?!ility. IfVc of both fresh and 24 hours in .ubat ·d
SS reel blood cells showed a shift to the left.IS compared to AA and AS red cells,"Ig-gesting an increased resistance toh;poL mic N aCI solution. T .e mean osmotictragility values of fresh SS red cells from0<3% to 0.5% NaCI solution were- ignificantly less than the AA and AS redcells. The mean osmotic fragility values ofincubated SS red cells were alsosignificantly different than the A and AS
values. The mean osmotic fragility values
A decrease was observed in the osmoticfragility of fresh and 24 hours incubatedred cells of sickle cell disease patients ascompared to that of normal (AA) and sicklecell trait (AS) red blood cells. The loss ofphosphatidylcholine from the SS red cellsmay be a possible cause for the decreased
osmotic fragility of red cells (6). Franck et01 (1983) have reported such type of changein sickle erythrocytes (7). The fragilitydecrease in sickle red cells may be dueto the greater loss of potassium from thered cells (5). A similar trend has beenreported in red cells of thalassaemiapatients (1),
Indian J Physiol Pharmacol 1999; 43(2) Letter to the Editor 269
B. P. DASH*, A. MITTRA** AND B. C. KAR***
*Regional Medical Research Centre (l.C.M.R.),Chandasekharpur,
Bhubaneswar - 751 016
**School of Life Sciences,Sambalpur University, Jyoti Vihar,
Burla - 768 019
and
'''''Y'''''Medical Aid and Reserch Centre,Burla - 768 017
REFERENCES
1. Dacie JV, Lewis SM. In Practical haematology(7th Ed), Churchill Livingstone, Edinburg, 1991:195-201.
2. Harris JW, Brewster HH, Ham TH, Castle WB.Studies on the destruction of red blood cells. X.The biophysics and biology of aickle cell disease.Arc Intern Med 1956; 97: 145-168.
3. Kulozik AE, Wainscoat JS, Serjeant GR, Kar BC,AI-Awamy B, Essam GJF, Falusi AG, Haque SK,Hilali AN, Kate S, Ranasinghe WAE, WeatherallDJ. Geographical survey of B-globin genehaplotypes: Evidence for an independent Asianorigin of the sickle cell mutation. Am J Hum Genet1986; 39: 239-244.
4. Dash BP, Mittra A, Kar BC. A study on the glucoseuptake, pyruvate and lactate formation in red blood
'Corresponding Author
cells of normal, sickle cell trait and sickle cellpatients. Indian J Clin Bi()chem 1992; 7:134-137.
5. Dash BP. Study of glucose metabolism in sicklehaemoglobin containing human red blood cells.Ph.D. Thesis, Sambalpur University, Orissa, India,1994. 100-102.
6. Kuypers FA, Chiu D, !Vlohandas N, RoelofsenB, Op den Kamp JAF, Lubin 13. The molecularspecies composition of phosphatidylcholine affectscellular properties in normal and sickelerythrocyts. Blood 1987; 70: 1111-1118.
7. Franck PF, Chiu D, Op den Kamp JAF, Van DeenenLLM, Roelofsen B. Accelerated transbilayermovement of phosphatidylcholine in sicklederythrocytes. A reversible process. J Bioi Chern1983; 258, 8435-8438.
