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Comparative estimation of hematocrit and trapped plasma in the packed cell volume in man, rabbit and chicken blood
(‘amp. Bwrhrm. Phniol. PrInted in Great Bnlain.
I to613,1981
Vol. 10A.pp.61 All rights reserved
CopyrIght
0300-962981 ‘I2061 I-03602 CWO 0 1981 Pergamon Press Ltd
COMPARATIVE ESTIMATION OF HEMATOCRIT AND TRAPPED PLASMA IN THE PACKED CELL VOLUME IN MAN, RABBIT AND CHICKEN BLOOD A. FERRANDO,I. G. BOBADILLA,M. S. BOBADILLA,A. PALOU and M. ALEMANY Bioquimica, Facultat de Citncies, Universitat de la Ciutat de Mallorca, Ciutat de Mallorca, Balears, Spain (Received 2 March 1981) Abstract-l. The effect of increasing centrifugation time and speed on hematocrit values of man, rabbit and chicken blood have been determined. 2. Plasma trapped volume in the cell pellet decreased with both speed and duration of centrifugation, remaining a significant value despite high time-speeds. 3. The volume of trapped plasma has been correlated with cell size, and cell shape affects the trapping of plasma more than cell size, as the avian blood cells were surrounded by comparatively larger amounts of plasma, under the same conditions, than the mammals.
INTRODUCTION The routine determination Wintrobe tubes (Wintrobe,
of hematocrit using the 1929) has been superseded
by micro-hematocrit determination in capillary tubes (Guest & Siler, 1934). This results in packed cell volumes which contain variable amounts of trapped plasma between the cells, due to the stability of the
cell form and the geometric constrictions of packaging stacks of non-adhering elastic cells (Lawson, 1962). The presence of trapped plasma in cell pellets has been sparsely acknowledged and studied (Chapin & Ross, 1942; Chaplin & Mollison, 1952; Hlad & Holmes, 1953; Ebaugh et al., 1955; Everett et al., 1956). However, the simple hematocrit value is sufficient for routine clinical and analytical uses. Little attention has been given to the variations of hematocrit with centrifugation time and speed (Lawson, 1962) and none at all to the estimation of trapped plasma in non-human species. We have ascertained the effects of time and speed of centrifugation upon hematocrit, and estimated the plasma trapped volume in a small red cell species (rabbit) and in a bird (chicken) with biconvex erythrocytes compared with human blood as control. MATERIALS AND METHODS Venous blood from voluntary healthy human male donors and blood obtained by decapitation of rabbits (Oryctolagus cuniculus) and chickens (Callus domesticus) were taken in dry-heparinized plastic tubes. Blood samples were used for cell counts with a Thoma grid dilution method. Two series of experiments were performed. In the first, blood samples in 100 mm heparinized capillary tubes were sealed with Cryto-Seal (Fisher) and sealing wax and centrifuged at 10°C for 5 min at different speeds in a swingout refrigerated centrifuge. The second experiment included the estimation of the hematocrit value in two series of tubes centrifuged during increasing periods of time at two given speeds: 900 and 3OOOrpm, corresponding to 131 9 and 14608 respectively (referred to a point 25 mm from the bottom of the blood column, of about 50 mm in 611
height). All centrifugal forces were referred to the same standard point and radius. The amount of plasma trapped in the packed cell volume was estimated by a dilution method based on the following manipulations. The whole blood was centrifuged 5 min at both 131 g and 14609. The plasma was used for protein determination (Lowry et al., 1951); the packed cell pellet in the tube was carefully washed with a small amount of saline (0.95%NaCl in water) which then was discarded. The original blood volume was then reconstituted with fresh saline and the precipitate carefully resuspended by slow rotation of the tube in a plane. When all cells were resuspended, the resulting fluid was centrifuged again under the same conditions as before. The protein content in the supernatant was then determined (Lowry et al., 1951). Possible hemolysis was checked through estimation of O.D. readings at 530 nm in both supernatants, as we found that hemolised blood cell supernatants had a high extinction coefficient at this wavelength. All hemolized samples were discarded. The calculation of trapped plasma volume TPV in the packed cell volume PCV was done as follows: TPV x PP = Spc. SPC x SV = TPV; ’ PP- SPC TPV + SV where PP was the plasma protein concentration, SPC the saline supernatant plasma concentration and SV the added saline volume. The TPV was calculated and used to determine the actual hematocrit value AHc from the apparent hematocrit Hc, estimated through centrifugation in a capillary tube at the same height as the original blood column and subjected to the same centrifugation pattern. Mean individual cell volume was estimated from the AHc and number of cells per liter (CN) values. The relationship between TPV and cell number and volume (CV) was calculated from: TPV 1 CN xcv x;;=cps: where CPS is the corrected plasma space referred to each cell. RESULTS AND DISCUSSION
Table 1 shows the apparent and calculated hematocrit values as well as cell counts and volumes, TPV
A. FERRANDOet al.
612
Table I. Hematic parameters of man, rabbit and chicken Parameter Hematocrit Hc (%) at 131 g at 14608 Cells per liter of blood CN (x 10’2) Plasma proteins PP (g/l) Trapped plasma volume TPV (7; of hematocrit) at 131 g at 146Og Mean corrected hematocrit value AHc (%) Mean individual cell volume CV(I) Plasma space in cell pellet CPS expressed as a % of cell volume (at 1460 g)
Human
Rabbit
Chicken
50.5 t 1.2 49.3 + 1.1
52.6 ) 1.1 36.4 f 0.8
60.4 + 1.4 41.7 * 1.1
4.53 f 0.78 68.3 rt 3.5
6.73 & 0.65 60.3 ) 1.4
3.28 f 0.22 73.4 + 4.1
16.8 &-3.1 6.9 + 1.1
33.9 f 1.4 8.9 f 1.2
34.2 & 2.7 16.4 i 1.3
41.6
38.8
34.8
91.8 x lo-l5
57.7 x lo-l5
106.1 x 1O-‘5
8.18
8.34
19.65
n = 5. Values = x f SE.
and CPS data for the three species studied. The mean hematocrit values were different at both speeds tested. The mean corrected AHc values for man and rabbit were not too much different. With comparable high speed TPV values however, the TPV value for chicken was comparatively higher with much increased CPS values. This could be due to the biconvex form of the nucleated avian erythrocyte compared with the better stackable biconcave mammalian red cells. In both the former mammals, each cell supported a mean 8% of their volume in plasma around them at 146Og, whereas this value was more than doubled for the chicken cells. The influence of cell volume on TPV seems to play only a minor role, as both human and fowl values were similar (with different TPV and CPS values), with similar data for rabbit and man despite the latter having a much higher mean cell size. The TPV values found here in human blood are in the same range as those previously reported by other authors (Gregersen & Shiro, 1937; Chapin & ROSS, 1942; Lawson, 1962). However, no comparable data for the other two species studied has been found in the literature. In Fig. 1 the effects of increasing centrifugal force on the apparent hematocrit values (Hcf are shown. The mean calculated AHc value has been indicated as a shaded area in all graphs; thus the clear space below the graph indicates the TPV and is a measure of the error introduced by differential spinning of the samples. The graphs become practically asymptotic at high g values; however in all cases Hc > AHc. These differences remain higher in the case of chicken. In Fig. 2 a similar graph is presented with varying centrifugation times. The effect of time of centrifugation seems to be higher in rabbit (probably because of the lower coefficient of sedimentation of smaller cells) and chicken (maybe due to its different cell shape) than in man, whose cells stack more easily. At higher speeds, however, the effects were considerably minimized, especially with longer centrifugation times. It must be pointed out, however, that despite the long
(30min) centrifugation, the relationship Hc > AHc was significantly maintained, suggesting there is a minimum TPV value for each cell size and shape. The differences between mammalian and avian CPS values suggest that care must be taken in the interpre-
Man
Hc
Rabbit
Chicken
0 0
500 Centrifugation
1000
IS00
force,g
Fig. 1. Relationship between apparent hematocrit (Hc), in %, and centrifugation force, in g, for human, rabbit and chicken blood. The shaded area corresponds to the real cell volume excluded TPV (AH@.
Hematocrit and trapped plasma in blood 100
r
6I3
tation of comparative hematocrit data for birds and mammals, as cell size, and especially cell shape. can alter the real cell size readings considerably, from apparent hematocrit and cell counts alone.
Man
REFERENCES
CHAPIN M. A. & Ross J. F. (1942) The determination
of the true ceil volume of dye dilution, by protein dilutions and with radioactive iron. The error of the centrifuge bematocrit. Am. J. Physiol. 137,447-453. CHAPLIN H. & MOLLISONP. L. (1952) Correction for plasma trapped in the red cell column of the hematocrit.
Rabbit
l
Hc
\
.
Blood I, 1227-1232.
Chicken ‘IC-0
50
0 0
IO
Centrifugation
20
30
time,min
Fig. 2. Relationships between apparent hematocrit (He), in %, and centrifugation time, in minutes, at 131 g (solid line, black dots) and at 146Og (dashed line, open dots). The shaded area corresponds to the real cell volume excluded TPV (AHc).
EBAUGHF. G., LEVINEP. & EMERSONC. P. (1955) The amount of trapped plasma in the red cell mass of the hematocrit tube. J. Lob. clin. Med. 46, 409-415. EVERETTN. B., SIMMONS B. & LASHERE. P. (1956) Distribution of blood (Fe?‘) and plasma (I”‘) volumes of rats determined by liquid nitrogen freezing. Cjrc~~afion Res. 4, 419-424. GREGERSEN M. I. & SCHRIOH. (1937) The behavior of the dye T-1824 with respect to its absorption by red blood cells and its fate in blood undergoing coagulation. Am. J. Physiol. 121, 284-291. GUEST G. M. & SILERV. E. (1934) A centrifuge method for the determination of the volume of cells in blood. J. Luh. c&n. Med. 19, 757-767. HLAD C. J. & HOLMESJ. Ii. (1953) Factors affecting hemat~rit determinations: trapped plasma, its amount and distribution. J. uppi. Physiol. 5, 457-465. LAWSON H. C. (1962) The volume of blood-a critical examination of methods for its measurement. In Handbook of Physiology, Section 2, Circulation, Vol. 1, pp. 23-49. American Phvsioloeical Societv. Bethesda. MD. LOWRY0. H., R~~EBR&JGH &. J., FARR~~. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. WINTROBE M. M. (1929) A simple and accurate hematocrit. J. Lab. c&n. Med. 15, 287-295.
I to613,1981
Vol. 10A.pp.61 All rights reserved
CopyrIght
0300-962981 ‘I2061 I-03602 CWO 0 1981 Pergamon Press Ltd
COMPARATIVE ESTIMATION OF HEMATOCRIT AND TRAPPED PLASMA IN THE PACKED CELL VOLUME IN MAN, RABBIT AND CHICKEN BLOOD A. FERRANDO,I. G. BOBADILLA,M. S. BOBADILLA,A. PALOU and M. ALEMANY Bioquimica, Facultat de Citncies, Universitat de la Ciutat de Mallorca, Ciutat de Mallorca, Balears, Spain (Received 2 March 1981) Abstract-l. The effect of increasing centrifugation time and speed on hematocrit values of man, rabbit and chicken blood have been determined. 2. Plasma trapped volume in the cell pellet decreased with both speed and duration of centrifugation, remaining a significant value despite high time-speeds. 3. The volume of trapped plasma has been correlated with cell size, and cell shape affects the trapping of plasma more than cell size, as the avian blood cells were surrounded by comparatively larger amounts of plasma, under the same conditions, than the mammals.
INTRODUCTION The routine determination Wintrobe tubes (Wintrobe,
of hematocrit using the 1929) has been superseded
by micro-hematocrit determination in capillary tubes (Guest & Siler, 1934). This results in packed cell volumes which contain variable amounts of trapped plasma between the cells, due to the stability of the
cell form and the geometric constrictions of packaging stacks of non-adhering elastic cells (Lawson, 1962). The presence of trapped plasma in cell pellets has been sparsely acknowledged and studied (Chapin & Ross, 1942; Chaplin & Mollison, 1952; Hlad & Holmes, 1953; Ebaugh et al., 1955; Everett et al., 1956). However, the simple hematocrit value is sufficient for routine clinical and analytical uses. Little attention has been given to the variations of hematocrit with centrifugation time and speed (Lawson, 1962) and none at all to the estimation of trapped plasma in non-human species. We have ascertained the effects of time and speed of centrifugation upon hematocrit, and estimated the plasma trapped volume in a small red cell species (rabbit) and in a bird (chicken) with biconvex erythrocytes compared with human blood as control. MATERIALS AND METHODS Venous blood from voluntary healthy human male donors and blood obtained by decapitation of rabbits (Oryctolagus cuniculus) and chickens (Callus domesticus) were taken in dry-heparinized plastic tubes. Blood samples were used for cell counts with a Thoma grid dilution method. Two series of experiments were performed. In the first, blood samples in 100 mm heparinized capillary tubes were sealed with Cryto-Seal (Fisher) and sealing wax and centrifuged at 10°C for 5 min at different speeds in a swingout refrigerated centrifuge. The second experiment included the estimation of the hematocrit value in two series of tubes centrifuged during increasing periods of time at two given speeds: 900 and 3OOOrpm, corresponding to 131 9 and 14608 respectively (referred to a point 25 mm from the bottom of the blood column, of about 50 mm in 611
height). All centrifugal forces were referred to the same standard point and radius. The amount of plasma trapped in the packed cell volume was estimated by a dilution method based on the following manipulations. The whole blood was centrifuged 5 min at both 131 g and 14609. The plasma was used for protein determination (Lowry et al., 1951); the packed cell pellet in the tube was carefully washed with a small amount of saline (0.95%NaCl in water) which then was discarded. The original blood volume was then reconstituted with fresh saline and the precipitate carefully resuspended by slow rotation of the tube in a plane. When all cells were resuspended, the resulting fluid was centrifuged again under the same conditions as before. The protein content in the supernatant was then determined (Lowry et al., 1951). Possible hemolysis was checked through estimation of O.D. readings at 530 nm in both supernatants, as we found that hemolised blood cell supernatants had a high extinction coefficient at this wavelength. All hemolized samples were discarded. The calculation of trapped plasma volume TPV in the packed cell volume PCV was done as follows: TPV x PP = Spc. SPC x SV = TPV; ’ PP- SPC TPV + SV where PP was the plasma protein concentration, SPC the saline supernatant plasma concentration and SV the added saline volume. The TPV was calculated and used to determine the actual hematocrit value AHc from the apparent hematocrit Hc, estimated through centrifugation in a capillary tube at the same height as the original blood column and subjected to the same centrifugation pattern. Mean individual cell volume was estimated from the AHc and number of cells per liter (CN) values. The relationship between TPV and cell number and volume (CV) was calculated from: TPV 1 CN xcv x;;=cps: where CPS is the corrected plasma space referred to each cell. RESULTS AND DISCUSSION
Table 1 shows the apparent and calculated hematocrit values as well as cell counts and volumes, TPV
A. FERRANDOet al.
612
Table I. Hematic parameters of man, rabbit and chicken Parameter Hematocrit Hc (%) at 131 g at 14608 Cells per liter of blood CN (x 10’2) Plasma proteins PP (g/l) Trapped plasma volume TPV (7; of hematocrit) at 131 g at 146Og Mean corrected hematocrit value AHc (%) Mean individual cell volume CV(I) Plasma space in cell pellet CPS expressed as a % of cell volume (at 1460 g)
Human
Rabbit
Chicken
50.5 t 1.2 49.3 + 1.1
52.6 ) 1.1 36.4 f 0.8
60.4 + 1.4 41.7 * 1.1
4.53 f 0.78 68.3 rt 3.5
6.73 & 0.65 60.3 ) 1.4
3.28 f 0.22 73.4 + 4.1
16.8 &-3.1 6.9 + 1.1
33.9 f 1.4 8.9 f 1.2
34.2 & 2.7 16.4 i 1.3
41.6
38.8
34.8
91.8 x lo-l5
57.7 x lo-l5
106.1 x 1O-‘5
8.18
8.34
19.65
n = 5. Values = x f SE.
and CPS data for the three species studied. The mean hematocrit values were different at both speeds tested. The mean corrected AHc values for man and rabbit were not too much different. With comparable high speed TPV values however, the TPV value for chicken was comparatively higher with much increased CPS values. This could be due to the biconvex form of the nucleated avian erythrocyte compared with the better stackable biconcave mammalian red cells. In both the former mammals, each cell supported a mean 8% of their volume in plasma around them at 146Og, whereas this value was more than doubled for the chicken cells. The influence of cell volume on TPV seems to play only a minor role, as both human and fowl values were similar (with different TPV and CPS values), with similar data for rabbit and man despite the latter having a much higher mean cell size. The TPV values found here in human blood are in the same range as those previously reported by other authors (Gregersen & Shiro, 1937; Chapin & ROSS, 1942; Lawson, 1962). However, no comparable data for the other two species studied has been found in the literature. In Fig. 1 the effects of increasing centrifugal force on the apparent hematocrit values (Hcf are shown. The mean calculated AHc value has been indicated as a shaded area in all graphs; thus the clear space below the graph indicates the TPV and is a measure of the error introduced by differential spinning of the samples. The graphs become practically asymptotic at high g values; however in all cases Hc > AHc. These differences remain higher in the case of chicken. In Fig. 2 a similar graph is presented with varying centrifugation times. The effect of time of centrifugation seems to be higher in rabbit (probably because of the lower coefficient of sedimentation of smaller cells) and chicken (maybe due to its different cell shape) than in man, whose cells stack more easily. At higher speeds, however, the effects were considerably minimized, especially with longer centrifugation times. It must be pointed out, however, that despite the long
(30min) centrifugation, the relationship Hc > AHc was significantly maintained, suggesting there is a minimum TPV value for each cell size and shape. The differences between mammalian and avian CPS values suggest that care must be taken in the interpre-
Man
Hc
Rabbit
Chicken
0 0
500 Centrifugation
1000
IS00
force,g
Fig. 1. Relationship between apparent hematocrit (Hc), in %, and centrifugation force, in g, for human, rabbit and chicken blood. The shaded area corresponds to the real cell volume excluded TPV (AH@.
Hematocrit and trapped plasma in blood 100
r
6I3
tation of comparative hematocrit data for birds and mammals, as cell size, and especially cell shape. can alter the real cell size readings considerably, from apparent hematocrit and cell counts alone.
Man
REFERENCES
CHAPIN M. A. & Ross J. F. (1942) The determination
of the true ceil volume of dye dilution, by protein dilutions and with radioactive iron. The error of the centrifuge bematocrit. Am. J. Physiol. 137,447-453. CHAPLIN H. & MOLLISONP. L. (1952) Correction for plasma trapped in the red cell column of the hematocrit.
Rabbit
l
Hc
\
.
Blood I, 1227-1232.
Chicken ‘IC-0
50
0 0
IO
Centrifugation
20
30
time,min
Fig. 2. Relationships between apparent hematocrit (He), in %, and centrifugation time, in minutes, at 131 g (solid line, black dots) and at 146Og (dashed line, open dots). The shaded area corresponds to the real cell volume excluded TPV (AHc).
EBAUGHF. G., LEVINEP. & EMERSONC. P. (1955) The amount of trapped plasma in the red cell mass of the hematocrit tube. J. Lob. clin. Med. 46, 409-415. EVERETTN. B., SIMMONS B. & LASHERE. P. (1956) Distribution of blood (Fe?‘) and plasma (I”‘) volumes of rats determined by liquid nitrogen freezing. Cjrc~~afion Res. 4, 419-424. GREGERSEN M. I. & SCHRIOH. (1937) The behavior of the dye T-1824 with respect to its absorption by red blood cells and its fate in blood undergoing coagulation. Am. J. Physiol. 121, 284-291. GUEST G. M. & SILERV. E. (1934) A centrifuge method for the determination of the volume of cells in blood. J. Luh. c&n. Med. 19, 757-767. HLAD C. J. & HOLMESJ. Ii. (1953) Factors affecting hemat~rit determinations: trapped plasma, its amount and distribution. J. uppi. Physiol. 5, 457-465. LAWSON H. C. (1962) The volume of blood-a critical examination of methods for its measurement. In Handbook of Physiology, Section 2, Circulation, Vol. 1, pp. 23-49. American Phvsioloeical Societv. Bethesda. MD. LOWRY0. H., R~~EBR&JGH &. J., FARR~~. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. WINTROBE M. M. (1929) A simple and accurate hematocrit. J. Lab. c&n. Med. 15, 287-295.