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Sickle-cell erythrocytes in the mongoose Herpestes sanguineus
180 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE.
Vol. 61.
No. 2.
1967.
COMMUNICATIONS SICKLE-CELL ERYTHROCYTES IN THE M O N G O O S E HERPESTES
SANGUINEUS BY
C. M. HAWKEY Nu~eld Institute of Comparative Medicine, The Zoological Society of London, N.W.1 AND
P. JORDAN Research and Control Department, Castries, St. Lucia, West Indies (Formerly at E.A. Institute for Medical Research, Mwanza, Tanzania) While examining blood samples from various animals for the presence of parasites, one of us (P.J.) noticed that the red cells of mongooses of the species Herpestes sanguineus (slender mongoose) were often sickle-shaped. Because of similarities between these cells and deoxygenated red cells of man suffering from sickle cell anaemia, the phenomenon was investigated to determine the conditions governing sickle cell formation in H. sanguineus. Two H. urva (crab-eating mongoose) were also examined to find out if sickling was a general property of erythrocytes of all mongooses. Blood was obtained from 6 H. sanguineus and 2 H. urva by puncture of a jugular or saphenous vein. Before venepuncture each animal received an intramuscular injection of phencyclidine hydrochloride (Sernylan, Parke Davis, 1.5 rag. per kg.) or nembutal intraperitoneally (60 mg. per kg.). Blood films and wet preparations were made directly from the syringe, and the remainder of the blood was mixed with anticoagulant as indicated. In all animals, red cells were present as normal biconcave discs when the blood was first obtained. Under certain circumstances many of the red cells of each H. sanguineus lost this normal shape and became elongated with sharply pointed ends. These cells, although similar in many ways to human sickle cells and sickle cells of the fallow deer, differed from them in that they were rarely curved. In many cells, more than 2 projections were present (Figure). Sickle cells were formed in blood mixed with the potassium salt of ethylenediamine tetra-acetic acid as anticoagulant (1.5 rag. E D T A per ml. of blood) and rotated on a Matburn mixer. Sickled red cells were also found in blood films allowed to dry slowly in air and in wet preparations diluted with 0.9% NaC1 and left overnight at 37°C. Spindling in the latter case was particularly marked near the edges of the coverslip. Changes in shape did not take place over a period of 24 hours at 4°C., 20°C. or 37°C. in sealed wet preparations of whole blood, wet preparations diluted with 2% sodium metabisulphite, heparinized, citrated or clotted blood or blood through which O., or CO., was bubbled. Alteration of the pH by one unit by addition of HC1 or NaOH did not have any effect. Cells which had already become sickled by mixture with EDTA immediately regained their normal shape when mixed with citrated plasma, serum or 0.9% NaC1, but not when subjected to EDTA plasma, 1-5% NaCI or 1-5% KC1 solutions. Bubbling CO., or O., through blood in which the cells were sickled had no effect.
(cl
(4 (a) (b) (c) (d)
Blood films prepared from H. sanguineus blood mixed with EDTA H. sanguineus blood mixed with 0.97’; NaCl H. sanguineus blood mixed with 1.20/ NaCl Oxygenated blood from fallow deer, Dama dama
181
C. M. HAWKEY AND P. JORDAN
These results suggested that sickling of the red cells was not related to oxygen tension but occurred when the salt concentration of the surrounding medium was increased. This was confirmed by suspending red cells in increasing concentrations of NaC1 or KC1. Solutions of 1~o and stronger caused rapid sickling of the red cells of H. sanguineus, whereas red cells of H. urva became crenated in the usual way. In all cases cells regained their normal shape when the salt concentration was reduced. The tendency for red cells to form sickles was found in each of the 6 H. sanguineus tested. Sickling did not take place in 2 H. urva. Red cell osmotic fragility was similar in H. sanguineus and the H. urva. Paper electrophoresis at p H 8.6 was carried out on haemolysates from H. sanguineus (LEHMANNand AGER, 1965). The haemoglobin moved as a single band to a position between human Hb-A and human Hb-S. Red cell and plasma Na ~ and K t estimations (by courtesy of Professor M. Maizells, University College Hospital, London) indicated that the red cells of H. sanguineus, like those of other Carnivora, do not possess the ability to concentrate these ions (Table). Full blood TABLE. Sodium and potassium levels in blood of H. sanguineus. Red cells
Plasma
Na +
K÷
Na T
K
H. sanguineus I
5.3
111
4"8
147
H. sanguineus H
6.1
108-5
5.3
147
Results in m-equiv, per litre. counts revealed no haematological abnormalities apart from 2 mongooses which were found to be infected with Monnigofilaria setanosei. Haemoglobin levels ranged from 14.4 to 15.4 g. per 100 ml. Elongation or sickling of red cells is known to occur in man with sickle cell disease and in all species of deer (GULLIVER, 1840). Erythrocytes of racoons, hamsters and squirrels can be induced to take up abnormal shapes, including some sickle-like forms, by prolonged storage or by treatment with hypertonic solutions, but this is considered to be of little significance by KITCHEN, PUTNAM and TAYLOR (1964) as the condition is irreversible. In human sickle cell anaemia, change in shape of the red cells is caused by the presence of haemoglobin S which forms insoluble elongated tactoids when in a reduced state (PERUTZand MITCHISON, 1950). Sickling of deer cells occurs in conditions of high oxygen concentration at alkaline pH, and is not associated with any pathological findings (KITCHEN, PUTNAM and TAYLOR, 1964). Our experiments on H. sanguineus suggest that red cells of this species contain haemoglobin of a type which is insoluble in situations of high salt concentration and is not influenced by oxygen tension. It is unlikely that the sickling tendency has any pathological significance in mongooses, as conditions under which it occurs could not exist in vivo. All the animals which we have examined have been in good health, with normal levels of haemoglobin. REFERENCES
GULLIVER,G. (1840). Proc. roy. Soc. Lond., IV, 199. LEHMANN, H. & AGER, J. (1961). Ass. Clin. Path. Broadsheet, No. 33. PERUTZ, M. F. & MITCHISON,J. M. (1950). Nature, Lond., 166, 677. KITCHEN, H., PUTNAM, F. & TAYLOR, W. (1964). Science, 144, 1237.
Vol. 61.
No. 2.
1967.
COMMUNICATIONS SICKLE-CELL ERYTHROCYTES IN THE M O N G O O S E HERPESTES
SANGUINEUS BY
C. M. HAWKEY Nu~eld Institute of Comparative Medicine, The Zoological Society of London, N.W.1 AND
P. JORDAN Research and Control Department, Castries, St. Lucia, West Indies (Formerly at E.A. Institute for Medical Research, Mwanza, Tanzania) While examining blood samples from various animals for the presence of parasites, one of us (P.J.) noticed that the red cells of mongooses of the species Herpestes sanguineus (slender mongoose) were often sickle-shaped. Because of similarities between these cells and deoxygenated red cells of man suffering from sickle cell anaemia, the phenomenon was investigated to determine the conditions governing sickle cell formation in H. sanguineus. Two H. urva (crab-eating mongoose) were also examined to find out if sickling was a general property of erythrocytes of all mongooses. Blood was obtained from 6 H. sanguineus and 2 H. urva by puncture of a jugular or saphenous vein. Before venepuncture each animal received an intramuscular injection of phencyclidine hydrochloride (Sernylan, Parke Davis, 1.5 rag. per kg.) or nembutal intraperitoneally (60 mg. per kg.). Blood films and wet preparations were made directly from the syringe, and the remainder of the blood was mixed with anticoagulant as indicated. In all animals, red cells were present as normal biconcave discs when the blood was first obtained. Under certain circumstances many of the red cells of each H. sanguineus lost this normal shape and became elongated with sharply pointed ends. These cells, although similar in many ways to human sickle cells and sickle cells of the fallow deer, differed from them in that they were rarely curved. In many cells, more than 2 projections were present (Figure). Sickle cells were formed in blood mixed with the potassium salt of ethylenediamine tetra-acetic acid as anticoagulant (1.5 rag. E D T A per ml. of blood) and rotated on a Matburn mixer. Sickled red cells were also found in blood films allowed to dry slowly in air and in wet preparations diluted with 0.9% NaC1 and left overnight at 37°C. Spindling in the latter case was particularly marked near the edges of the coverslip. Changes in shape did not take place over a period of 24 hours at 4°C., 20°C. or 37°C. in sealed wet preparations of whole blood, wet preparations diluted with 2% sodium metabisulphite, heparinized, citrated or clotted blood or blood through which O., or CO., was bubbled. Alteration of the pH by one unit by addition of HC1 or NaOH did not have any effect. Cells which had already become sickled by mixture with EDTA immediately regained their normal shape when mixed with citrated plasma, serum or 0.9% NaC1, but not when subjected to EDTA plasma, 1-5% NaCI or 1-5% KC1 solutions. Bubbling CO., or O., through blood in which the cells were sickled had no effect.
(cl
(4 (a) (b) (c) (d)
Blood films prepared from H. sanguineus blood mixed with EDTA H. sanguineus blood mixed with 0.97’; NaCl H. sanguineus blood mixed with 1.20/ NaCl Oxygenated blood from fallow deer, Dama dama
181
C. M. HAWKEY AND P. JORDAN
These results suggested that sickling of the red cells was not related to oxygen tension but occurred when the salt concentration of the surrounding medium was increased. This was confirmed by suspending red cells in increasing concentrations of NaC1 or KC1. Solutions of 1~o and stronger caused rapid sickling of the red cells of H. sanguineus, whereas red cells of H. urva became crenated in the usual way. In all cases cells regained their normal shape when the salt concentration was reduced. The tendency for red cells to form sickles was found in each of the 6 H. sanguineus tested. Sickling did not take place in 2 H. urva. Red cell osmotic fragility was similar in H. sanguineus and the H. urva. Paper electrophoresis at p H 8.6 was carried out on haemolysates from H. sanguineus (LEHMANNand AGER, 1965). The haemoglobin moved as a single band to a position between human Hb-A and human Hb-S. Red cell and plasma Na ~ and K t estimations (by courtesy of Professor M. Maizells, University College Hospital, London) indicated that the red cells of H. sanguineus, like those of other Carnivora, do not possess the ability to concentrate these ions (Table). Full blood TABLE. Sodium and potassium levels in blood of H. sanguineus. Red cells
Plasma
Na +
K÷
Na T
K
H. sanguineus I
5.3
111
4"8
147
H. sanguineus H
6.1
108-5
5.3
147
Results in m-equiv, per litre. counts revealed no haematological abnormalities apart from 2 mongooses which were found to be infected with Monnigofilaria setanosei. Haemoglobin levels ranged from 14.4 to 15.4 g. per 100 ml. Elongation or sickling of red cells is known to occur in man with sickle cell disease and in all species of deer (GULLIVER, 1840). Erythrocytes of racoons, hamsters and squirrels can be induced to take up abnormal shapes, including some sickle-like forms, by prolonged storage or by treatment with hypertonic solutions, but this is considered to be of little significance by KITCHEN, PUTNAM and TAYLOR (1964) as the condition is irreversible. In human sickle cell anaemia, change in shape of the red cells is caused by the presence of haemoglobin S which forms insoluble elongated tactoids when in a reduced state (PERUTZand MITCHISON, 1950). Sickling of deer cells occurs in conditions of high oxygen concentration at alkaline pH, and is not associated with any pathological findings (KITCHEN, PUTNAM and TAYLOR, 1964). Our experiments on H. sanguineus suggest that red cells of this species contain haemoglobin of a type which is insoluble in situations of high salt concentration and is not influenced by oxygen tension. It is unlikely that the sickling tendency has any pathological significance in mongooses, as conditions under which it occurs could not exist in vivo. All the animals which we have examined have been in good health, with normal levels of haemoglobin. REFERENCES
GULLIVER,G. (1840). Proc. roy. Soc. Lond., IV, 199. LEHMANN, H. & AGER, J. (1961). Ass. Clin. Path. Broadsheet, No. 33. PERUTZ, M. F. & MITCHISON,J. M. (1950). Nature, Lond., 166, 677. KITCHEN, H., PUTNAM, F. & TAYLOR, W. (1964). Science, 144, 1237.