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Transient velocity sedimentation at unit gravity of human erythrocytes
ANALYTICAL
BIOCHEMISTRY
81,
143-150 (1977)
Transient Velocity Sedimentation at Unit Gravity of Human Erythrocytes S. N. AGATHOS,
A. L. GRIFFITH, R. UAUY-DAGACH, AND N. CATSIMPOOLAS’
V. R. YOUNG,
Department of Nlrtririon und Food Science, Massuchusetts Instit:rte of Technology. Cambridge, Massachlcserts 02139 Received December 6. 1976: accepted April 4. 1977 A new biophysical method, transient velocity sedimentation (TRANSVELS) at unit gravity, has been applied to the study of human erythrocytes from normal subjects and from patients with diseased states producing abnormal red blood cells. The hydrodynamic behavior of the cells during sedimentation was examined by repetitively recording the distribution profile as a function of time by the use of optical scanning methods. Computer analysis of the distributions allowed precise measurement of the mean sedimentation velocity (S) and the mean skewness (+s ) of peak profile. A two-dimensional plot of s versus 3 indicated significant differences between ” normal” and “abnormal” erythrocytes by linear categorization techniques and probability density ellipses at the 0.1 density level. It is concluded that precise measurement of these two new parameters offers a new and valuable approach to the physical characterization of erythrocytes.
We have recently described a new instrumentation system (1) and procedures (2) for the kinetic analysis of the sedimentation of mammalian cells at unit gravity (lg). The transient velocity sedimentation (TRANSVELS) technique provides precise measurement of the mean sedimentation velocity ( S) and the mean skewness ( 3) of each cell population. This type of biophysical analysis can be performed with as few as 0.5 x IO” cells, thereby facilitating studies in which a small biological sample is available. Since the sedimentation of cells at Ig depends on a number of factors including cell volume, density, shape, and, probably, surface deformability and texture, we have attempted to apply the TRANSVELS method to examine the hydrodynamic behavior of normal and abnormal erythrocytes. This was not intended to be a major clinical study, but rather a search for conditions under which the technique could be tested for its sensitivity in detecting cell alterations induced by disease. We, therefore, report in this paper some encouraging preliminary studies which will be further explored and refined in the near future. ’ To whom requests for reprints should be addressed
ISSN 0003-2697
144
AGATHOS
MATERIALS
ET AL.
AND METHODS
The blood samples used for these studies were obtained from normal subjects and from patients with spherocytosis, elliptocytosis, sickle cell SC, sickle cell SS, a-thalassemia, and red blood cell aplasia (See Table 1). The blood cells were suspended in Alsever’s solution and were washed twice with phosphate-buffered saline (PBS) before analysis. The instrument used for these experiments has been described in detail elsewhere (1,3). Optical scanning was performed at 415 nm. Sedimentation was carried out in a l-2% bovine serum albumin (BSA) gradient in phosphate-buffered saline (PBS) at 25°C as described previously. The mean sedimentation velocity (S in centimeters per second) was estimated by linear regression analysis of the change of the first statistical moment (m’,) of the cell distribution as a function of sedimentation time (t); therefore, S = dm’,/dt (2). The mean skewness (3) was estimated statistically from the skewness (S) values of each cell distribution at different times (t,) of sedimentation. S values were computed from the second (mJ and third (m,) central statistical moments of each cell distribution by (3) S = m,lm2)3’2. The estimation of HZ’,, m,, and trz3 by computer has been described in detail elsewhere (3). The mean skew-
FIG. 1. Typical TRANS-VELS profiles of erythrocytes and from a patient with cu-thalassemia. Superimposed scans sedimentation are shown for comparison.
from an elderly male of the two distributions
subject during
SEDIMENTATION
OF ERYTHROCYTES
T,rnP
t5ri
145
* -8 3l
FIG. 2. Plot of peak position (mean, first statistical moment) as a function of sedimentation time for the cell populations shown in Fig. 1. The slope of the lines corresponds numerically to the mean sedimentation velocity ( 3).
ness 3 provides a measure of peak related primarily to cell heterogeneity in the unit gravity field. RESULTS
assymetry in regard
which. physically, is to transport behavior
AND DISCUSSION
A typical TRANS-VELS pattern of normal RBC from an elderly male and of RBC from an a-thalassemia patient is shown in Fig. 1. It is evident from the diagram that normal RBC sediment at a considerably faster rate in the BSAiPBS gradient. The shape of the distribution is also different, which is reflected in the statistical measurement of the mean skewness (3). The plot of mean peak position (m’,) as a function of sedimentation time (Fig. 2) exhibited straight lines with correlation coefficients better than 0.9990. The slope of each line produced a numerical value for the mean sedimentation velocity ( 3) of the cells. A series of such experiments were performed with RBC from normal subjects (11 samples) and from patients with RBC abnormalities (8 samples). A two-dimensional plot of the experimentally obtained .? and 3
146
AGATHOS
ET AL.
values from the individual RBC populations, along with the mean values of the “normal” and “abnormal” group, is shown in Fig. 3. Confidence ellipses at the 0.1 probability density levels were estimated by computer for each group and are indicated by dotted lines in Fig. 3. In general, the “abnormal” group of RBC exhibited lower sedimentation velocity (S) values and lower ,!? values than did the “normal” group. Although the confidence ellipses were partially overlapping, the majority of the cell samples (90%) were not found in the common region. The significant difference between these groups of cells is also indicated by the fact that neither of the ellipses overlaps the center of the other. Furthermore, employment of linear categorization techniques (4) allowed the determination of a threshold value T and discriminator values D, for each cell population. A linear categorizer assigns an “unknown” cell population to a certain category when the cell data evaluation leads to a discriminator value D, less than a threshold T and to another category if D, is larger or equal to T. Table 1 shows the D, values obtained from S and s data for each population of cells by a computer program available in this laboratory. In all cases, the cell populations were assigned to the
t 2.0.
r
l-_
,x--
“NORMAL”
RBC
_
I t3t3-
, ;
*
lo lo x 22
'.6‘.6-
;
1.2-
, ,,&;& / \
I
i
-Is2,’
;?-5'\t;
0In I420 \
I o-
\
I
\
,
'; \
-\ -\ '-_ ‘-_
'_ -7-L -7-L
';
“ABNORMAL”
RBC
0.80.8-
0.6 0.6-
" -0.6
A
' -0.4I"
L
I -0.2
I'
0
'
" 0.2
"I 04
" I
0.6
I
0.0
' I
s FIG. 3. Two-dimensional plot of mean sedimentation velocity (S) values versus mean skewness (3) values for a group of erythrocytes from normal subjects and a group of patients exhibiting red blood cell abnormalities (i.e.. spherocytosis. a-thalassemia. sickle cell anemia and red blood cell aplasia). The dotted line indicates computer-estimated confidence ellipses for the two groups at the 0.1 probability density level. The solid line illustrates the threshold level T for linear categorization of the two groups of cells.
SEDIMENTATION
TABLE VALUES
OF S. 5 AND D, FOR “NORMAL”
1
AND “ABNORMAL”
Cell category “Normal” Sample No. I 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
’ M. male: a-thalassemia:
147
OF ERYTHROCYTES
HUMAN
RED BLOOD
CELLS
Cell category “Abnormal”
s x 10
s
D,”
1.70 1.80 1.54 I .59 1.64 1.55 1.67 1.65 I .65 1.75 1.54 1.63
0.48 0.59 0.49 0.51 0.51 0.19 0.22 0.21 0.42 0.39 0.24 0.30
1.58 1.73 1.47 1.52 1.56 1.28 1.39 1.37 1.51 1.56 I .30 1.41
i x 10’
D,”
Description” F F F F F M M
I .65 1.40 1.22 1.36 1.32 1.23 1.14 1.36
F, female; SPH. splerocytosis: APL. RBC aplasia: number
s
-0.01 0.00 -0.05 0.06 0.04 0.15 0.37 0.28
1.”-1.04 0.87 I .os 1.01 1.01 1.09 1.20
ELL. elliptocytosis; SC. sickle within parentheses is age.
(20) (20) (20) (33) (20) (68) (65) M (68) M (20) M (20) M (31) M (20) SPH. SPH. ELL. SC. F SC, F THA. THA. APL,
cell:
M (3) F (7) F (1) (27) (5) M (9) M (39) F (2)
THA.
correct group (i.e., normal and abnormal) by the linear categorizer. Therefore, we have concluded that the combined measurement of the S and s values by the TRANS-VELS technique offers two new valuable experimental parameters for the physical characterization of erythrocytes and other mammalian cells. Presently, we do not attempt to explain the possible causes for the observed differences among the cell populations examined. Changes in volume, density, and shape, as well as in heterogeneity of individual cells within a sample population (e.g., cY-thalassemia patient) in regard to these properties, may contribute to altered sedimentation behavior. Furthermore, physical alterations are the result of even more complex biochemical events. There is a need to examine a large number of RBC samples from healthy subjects, as a function of at least sex and age, to determine the normal dispersion of S and 3 values among various groups. In addition, a large number of patients with diagnosed red blood cell anomalies must
148
AGATHOS
ET AL.
be examined if biophysical criteria can be established for the presence of certain pathological RBC conditions. Furthermore, it will be interesting to know if changes in the hydrodynamic properties of RBC under lg sedimentation can be observed in cases of malnutrition and in other diseased states. At present, it appears that the TRANS-VELS technique offers a new tool for probing the physical characteristics of intact cells. APPENDIX Linear
Categorization
An unknown cell population is assigned by a linear categorizer to a when the cell data evalucertain category (e.g., “normal” or “abnormal”) ation leads to a descriptor value D, less than a threshold T. The D, and T values are estimated as follows. The mean sedimentation velocity (S) for a particular cell population is estimated from the slope of an m’, versus time plot (Fig. 2). The mean skewness (s) is estimated from the skewness (S) values obtained from each individual scan (Fig. 1). Next, the mean Si and 3, values of all cell populations of class I (e.g., “abnormal”) and Sri, s,, values of all cell populations of class II (e.g., “normal”) are determined. The .?r, s,, and S,,, s,, pair values determine the centroids of the distribution of each class of cells (see Fig. 3). Subsequently, the coordinates Xlr and XZT of a symmetrically placed boundary line between cell category centroids follow as:
X2T = (S,, + S,)/2. The weight coefficients ~1, and ~3~are estimated
by:
U’, = s,, - s, H’2 = s,, - 3,. Consequently,
the value T is given by:
T = Xlr The discriminator
h’q M’l + x27 + ),‘22)i i . (1.tl,2 + w2*p ( 11’,2 1 i i
value D, of a certain cell population
Ellipse
of a Bivariate
is computed from:
Distribution
The construction of the ellipse of a given probability density (q) of a bivariate distribution of values J: and s obtained from several cell populations is performed as follows.
SEDIMENTATION
149
OF ERYTHROCYTES
“Abnormal” cell populations are considered as class 1 and “normal” cell populations as class II. The variances of S and 3 values of class I are computed from:
a1
i=l
?=
II - 1
and
The covariance follows as: + co”
1.2 -
(ii
-
S,)(Si
-
3,)
‘=I
II - 1
The semiaxes of the wanted ellipse. a and b, are then given by: ‘, = K(a,’
+ u.‘2 + [(a,” ~ a,‘)’
+ 4 COV,,..2)~]+
and
where:
p denotes the correlation coefficient, and q the desired probability density level (e.g., 0.1). 6, the angle by which the ellipse is rotated, follows as: 8 = 55 tan-’ 2 The coordinates
COVI.:! ’ u.“l - u,2 i . i
of the ellipses vertices follow as: Major
axis: S, + n sir? 8. 3, + LZ cos 0 j I - CI sin 8. 3, - CI cos 0
Minor
axis: S, + h cos 8, 3, - h sin 0 s , -b cos 0.3, - b sin 0
The above computations are repeated populations belonging to class II.
for
the ellipse
of the cell
150
AGATHOS
ET AL.
ACKNOWLEDGMENTS This work was supported by NC1 Contract No. NOI-CB-43928 and NSF Grant No. MPS74- 19830. The technical assistance of Ms. Robin Rossi in some of these experiments is acknowledged with thanks. We are also grateful to Professor D. Nathan (Division of Hematology-Oncology. Children’s Hospital Medical Center. Boston. Mass.) for providing the erythrocyte samples from diseased states and for valuable review of this paper.
REFERENCES I. Catsimpoolas. N., Griffith. A. L.. Williams, J. M.. Chrambach. A., and Rodbard. D. (1975) Am/. Biochem. 69, 372-384. 2. Catsimpoolas, N.. Rossi, R., and Griffith, A. L. (1976) Life Sci. 18, 481-488. 3. Catsimpoolas. N. (1975) in Methods of Protein Separation (Catsimpoolas. N., ed.). Vol. 1, pp. 27-67. Plenum Press, New York. 4. Wied. G. L.. Bahr, G. F.. and Bartels. P. H. (1970) in Automated Cell Identihcation and Cell Sorting (Wied. G. L.. and Bahr. G. F., eds.), pp. 195-360. Academic Press, New York.
BIOCHEMISTRY
81,
143-150 (1977)
Transient Velocity Sedimentation at Unit Gravity of Human Erythrocytes S. N. AGATHOS,
A. L. GRIFFITH, R. UAUY-DAGACH, AND N. CATSIMPOOLAS’
V. R. YOUNG,
Department of Nlrtririon und Food Science, Massuchusetts Instit:rte of Technology. Cambridge, Massachlcserts 02139 Received December 6. 1976: accepted April 4. 1977 A new biophysical method, transient velocity sedimentation (TRANSVELS) at unit gravity, has been applied to the study of human erythrocytes from normal subjects and from patients with diseased states producing abnormal red blood cells. The hydrodynamic behavior of the cells during sedimentation was examined by repetitively recording the distribution profile as a function of time by the use of optical scanning methods. Computer analysis of the distributions allowed precise measurement of the mean sedimentation velocity (S) and the mean skewness (+s ) of peak profile. A two-dimensional plot of s versus 3 indicated significant differences between ” normal” and “abnormal” erythrocytes by linear categorization techniques and probability density ellipses at the 0.1 density level. It is concluded that precise measurement of these two new parameters offers a new and valuable approach to the physical characterization of erythrocytes.
We have recently described a new instrumentation system (1) and procedures (2) for the kinetic analysis of the sedimentation of mammalian cells at unit gravity (lg). The transient velocity sedimentation (TRANSVELS) technique provides precise measurement of the mean sedimentation velocity ( S) and the mean skewness ( 3) of each cell population. This type of biophysical analysis can be performed with as few as 0.5 x IO” cells, thereby facilitating studies in which a small biological sample is available. Since the sedimentation of cells at Ig depends on a number of factors including cell volume, density, shape, and, probably, surface deformability and texture, we have attempted to apply the TRANSVELS method to examine the hydrodynamic behavior of normal and abnormal erythrocytes. This was not intended to be a major clinical study, but rather a search for conditions under which the technique could be tested for its sensitivity in detecting cell alterations induced by disease. We, therefore, report in this paper some encouraging preliminary studies which will be further explored and refined in the near future. ’ To whom requests for reprints should be addressed
ISSN 0003-2697
144
AGATHOS
MATERIALS
ET AL.
AND METHODS
The blood samples used for these studies were obtained from normal subjects and from patients with spherocytosis, elliptocytosis, sickle cell SC, sickle cell SS, a-thalassemia, and red blood cell aplasia (See Table 1). The blood cells were suspended in Alsever’s solution and were washed twice with phosphate-buffered saline (PBS) before analysis. The instrument used for these experiments has been described in detail elsewhere (1,3). Optical scanning was performed at 415 nm. Sedimentation was carried out in a l-2% bovine serum albumin (BSA) gradient in phosphate-buffered saline (PBS) at 25°C as described previously. The mean sedimentation velocity (S in centimeters per second) was estimated by linear regression analysis of the change of the first statistical moment (m’,) of the cell distribution as a function of sedimentation time (t); therefore, S = dm’,/dt (2). The mean skewness (3) was estimated statistically from the skewness (S) values of each cell distribution at different times (t,) of sedimentation. S values were computed from the second (mJ and third (m,) central statistical moments of each cell distribution by (3) S = m,lm2)3’2. The estimation of HZ’,, m,, and trz3 by computer has been described in detail elsewhere (3). The mean skew-
FIG. 1. Typical TRANS-VELS profiles of erythrocytes and from a patient with cu-thalassemia. Superimposed scans sedimentation are shown for comparison.
from an elderly male of the two distributions
subject during
SEDIMENTATION
OF ERYTHROCYTES
T,rnP
t5ri
145
* -8 3l
FIG. 2. Plot of peak position (mean, first statistical moment) as a function of sedimentation time for the cell populations shown in Fig. 1. The slope of the lines corresponds numerically to the mean sedimentation velocity ( 3).
ness 3 provides a measure of peak related primarily to cell heterogeneity in the unit gravity field. RESULTS
assymetry in regard
which. physically, is to transport behavior
AND DISCUSSION
A typical TRANS-VELS pattern of normal RBC from an elderly male and of RBC from an a-thalassemia patient is shown in Fig. 1. It is evident from the diagram that normal RBC sediment at a considerably faster rate in the BSAiPBS gradient. The shape of the distribution is also different, which is reflected in the statistical measurement of the mean skewness (3). The plot of mean peak position (m’,) as a function of sedimentation time (Fig. 2) exhibited straight lines with correlation coefficients better than 0.9990. The slope of each line produced a numerical value for the mean sedimentation velocity ( 3) of the cells. A series of such experiments were performed with RBC from normal subjects (11 samples) and from patients with RBC abnormalities (8 samples). A two-dimensional plot of the experimentally obtained .? and 3
146
AGATHOS
ET AL.
values from the individual RBC populations, along with the mean values of the “normal” and “abnormal” group, is shown in Fig. 3. Confidence ellipses at the 0.1 probability density levels were estimated by computer for each group and are indicated by dotted lines in Fig. 3. In general, the “abnormal” group of RBC exhibited lower sedimentation velocity (S) values and lower ,!? values than did the “normal” group. Although the confidence ellipses were partially overlapping, the majority of the cell samples (90%) were not found in the common region. The significant difference between these groups of cells is also indicated by the fact that neither of the ellipses overlaps the center of the other. Furthermore, employment of linear categorization techniques (4) allowed the determination of a threshold value T and discriminator values D, for each cell population. A linear categorizer assigns an “unknown” cell population to a certain category when the cell data evaluation leads to a discriminator value D, less than a threshold T and to another category if D, is larger or equal to T. Table 1 shows the D, values obtained from S and s data for each population of cells by a computer program available in this laboratory. In all cases, the cell populations were assigned to the
t 2.0.
r
l-_
,x--
“NORMAL”
RBC
_
I t3t3-
, ;
*
lo lo x 22
'.6‘.6-
;
1.2-
, ,,&;& / \
I
i
-Is2,’
;?-5'\t;
0In I420 \
I o-
\
I
\
,
'; \
-\ -\ '-_ ‘-_
'_ -7-L -7-L
';
“ABNORMAL”
RBC
0.80.8-
0.6 0.6-
" -0.6
A
' -0.4I"
L
I -0.2
I'
0
'
" 0.2
"I 04
" I
0.6
I
0.0
' I
s FIG. 3. Two-dimensional plot of mean sedimentation velocity (S) values versus mean skewness (3) values for a group of erythrocytes from normal subjects and a group of patients exhibiting red blood cell abnormalities (i.e.. spherocytosis. a-thalassemia. sickle cell anemia and red blood cell aplasia). The dotted line indicates computer-estimated confidence ellipses for the two groups at the 0.1 probability density level. The solid line illustrates the threshold level T for linear categorization of the two groups of cells.
SEDIMENTATION
TABLE VALUES
OF S. 5 AND D, FOR “NORMAL”
1
AND “ABNORMAL”
Cell category “Normal” Sample No. I 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
’ M. male: a-thalassemia:
147
OF ERYTHROCYTES
HUMAN
RED BLOOD
CELLS
Cell category “Abnormal”
s x 10
s
D,”
1.70 1.80 1.54 I .59 1.64 1.55 1.67 1.65 I .65 1.75 1.54 1.63
0.48 0.59 0.49 0.51 0.51 0.19 0.22 0.21 0.42 0.39 0.24 0.30
1.58 1.73 1.47 1.52 1.56 1.28 1.39 1.37 1.51 1.56 I .30 1.41
i x 10’
D,”
Description” F F F F F M M
I .65 1.40 1.22 1.36 1.32 1.23 1.14 1.36
F, female; SPH. splerocytosis: APL. RBC aplasia: number
s
-0.01 0.00 -0.05 0.06 0.04 0.15 0.37 0.28
1.”-1.04 0.87 I .os 1.01 1.01 1.09 1.20
ELL. elliptocytosis; SC. sickle within parentheses is age.
(20) (20) (20) (33) (20) (68) (65) M (68) M (20) M (20) M (31) M (20) SPH. SPH. ELL. SC. F SC, F THA. THA. APL,
cell:
M (3) F (7) F (1) (27) (5) M (9) M (39) F (2)
THA.
correct group (i.e., normal and abnormal) by the linear categorizer. Therefore, we have concluded that the combined measurement of the S and s values by the TRANS-VELS technique offers two new valuable experimental parameters for the physical characterization of erythrocytes and other mammalian cells. Presently, we do not attempt to explain the possible causes for the observed differences among the cell populations examined. Changes in volume, density, and shape, as well as in heterogeneity of individual cells within a sample population (e.g., cY-thalassemia patient) in regard to these properties, may contribute to altered sedimentation behavior. Furthermore, physical alterations are the result of even more complex biochemical events. There is a need to examine a large number of RBC samples from healthy subjects, as a function of at least sex and age, to determine the normal dispersion of S and 3 values among various groups. In addition, a large number of patients with diagnosed red blood cell anomalies must
148
AGATHOS
ET AL.
be examined if biophysical criteria can be established for the presence of certain pathological RBC conditions. Furthermore, it will be interesting to know if changes in the hydrodynamic properties of RBC under lg sedimentation can be observed in cases of malnutrition and in other diseased states. At present, it appears that the TRANS-VELS technique offers a new tool for probing the physical characteristics of intact cells. APPENDIX Linear
Categorization
An unknown cell population is assigned by a linear categorizer to a when the cell data evalucertain category (e.g., “normal” or “abnormal”) ation leads to a descriptor value D, less than a threshold T. The D, and T values are estimated as follows. The mean sedimentation velocity (S) for a particular cell population is estimated from the slope of an m’, versus time plot (Fig. 2). The mean skewness (s) is estimated from the skewness (S) values obtained from each individual scan (Fig. 1). Next, the mean Si and 3, values of all cell populations of class I (e.g., “abnormal”) and Sri, s,, values of all cell populations of class II (e.g., “normal”) are determined. The .?r, s,, and S,,, s,, pair values determine the centroids of the distribution of each class of cells (see Fig. 3). Subsequently, the coordinates Xlr and XZT of a symmetrically placed boundary line between cell category centroids follow as:
X2T = (S,, + S,)/2. The weight coefficients ~1, and ~3~are estimated
by:
U’, = s,, - s, H’2 = s,, - 3,. Consequently,
the value T is given by:
T = Xlr The discriminator
h’q M’l + x27 + ),‘22)i i . (1.tl,2 + w2*p ( 11’,2 1 i i
value D, of a certain cell population
Ellipse
of a Bivariate
is computed from:
Distribution
The construction of the ellipse of a given probability density (q) of a bivariate distribution of values J: and s obtained from several cell populations is performed as follows.
SEDIMENTATION
149
OF ERYTHROCYTES
“Abnormal” cell populations are considered as class 1 and “normal” cell populations as class II. The variances of S and 3 values of class I are computed from:
a1
i=l
?=
II - 1
and
The covariance follows as: + co”
1.2 -
(ii
-
S,)(Si
-
3,)
‘=I
II - 1
The semiaxes of the wanted ellipse. a and b, are then given by: ‘, = K(a,’
+ u.‘2 + [(a,” ~ a,‘)’
+ 4 COV,,..2)~]+
and
where:
p denotes the correlation coefficient, and q the desired probability density level (e.g., 0.1). 6, the angle by which the ellipse is rotated, follows as: 8 = 55 tan-’ 2 The coordinates
COVI.:! ’ u.“l - u,2 i . i
of the ellipses vertices follow as: Major
axis: S, + n sir? 8. 3, + LZ cos 0 j I - CI sin 8. 3, - CI cos 0
Minor
axis: S, + h cos 8, 3, - h sin 0 s , -b cos 0.3, - b sin 0
The above computations are repeated populations belonging to class II.
for
the ellipse
of the cell
150
AGATHOS
ET AL.
ACKNOWLEDGMENTS This work was supported by NC1 Contract No. NOI-CB-43928 and NSF Grant No. MPS74- 19830. The technical assistance of Ms. Robin Rossi in some of these experiments is acknowledged with thanks. We are also grateful to Professor D. Nathan (Division of Hematology-Oncology. Children’s Hospital Medical Center. Boston. Mass.) for providing the erythrocyte samples from diseased states and for valuable review of this paper.
REFERENCES I. Catsimpoolas. N., Griffith. A. L.. Williams, J. M.. Chrambach. A., and Rodbard. D. (1975) Am/. Biochem. 69, 372-384. 2. Catsimpoolas, N.. Rossi, R., and Griffith, A. L. (1976) Life Sci. 18, 481-488. 3. Catsimpoolas. N. (1975) in Methods of Protein Separation (Catsimpoolas. N., ed.). Vol. 1, pp. 27-67. Plenum Press, New York. 4. Wied. G. L.. Bahr, G. F.. and Bartels. P. H. (1970) in Automated Cell Identihcation and Cell Sorting (Wied. G. L.. and Bahr. G. F., eds.), pp. 195-360. Academic Press, New York.