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The influence of low protein s plasma levels in young women, on the definition of normal range
Thrombosis Research. Vol. 73, No. 2. pp. 149-152, 1994 Copyright Q 1994 Elrevier Science Ltd Printed in the USA. AlI rights resetved 0049-3848/94 $6.00 + .@I
Pergamon
MINI REPORT
THE INFLUENCE OF LOW PROTEIN S PLASMA LEVELS IN YOUNG WOMEN, ON THE DEFINITION OF NORMAL RANGE.
M. Gari, L. Falkon, T. Urrutia, C. VallvC, M. Borrell, J. Fontcuberta. Unitat d’Hemost&ia i Trombosi. Hospital de la Santa Creu i Sant Pau. Barcelona, Spain.
(Received
15 April 1993 by Editor C. Soria; accepted 5 November
1993)
The normal range for plasma Protein S (PS) concentration using different methods has been reported by several investigators, and values show evident discrepancies (1,2,3). The reasons which might al&t these results are still unclear, although some reports show the influence of sex in plasma PS levels (4) and others had reported changes during pregnancy and in women on oral contraceptives (5,6). With the purpose to define the normal range for accurately diagnose deficient states (congenital and/or acquired), we study: l- the possible influence of sex and/or age in plasma PS levels (for three different methods) in a healthy population (n= 130) divided into 4 groups according to age and sex. 2- the relationship between immunological and functional methods for PS quantification.
SUBJECTS. MATERIALS AND METHODS 130 plasma samples were obtained from healthy blood donors and were used to establish the normal range and the relationship between methods. They were distributed into 4 groups: a) 28 males, mean age 53.6 years (range 46 to 65). b) 23 females, mean age 53.8 years (range 46 to 65). c) 38 males, mean age 32.6 years (range 18 to 45). d) 41 females, mean age 35.3 years (range 18 to 45). 46 different samples were obtained from non-anticoagulated individuals with low PS plasma levels, as detected by previous determinations. This group together with the 130 blood donors plasmas were used to study the relationship between methods. Following 12 hours’ fasting, samples were drawn between 9.00 and 11.00 am, by veinpuncture into vacutainer tubes containing 13 mM trisodum citrate. Platelet poor plasma (ppp) was prepared by centrifugation at 3,500 rpm for 20 minutes at room temperature. Plasma samples were stored at -40°C until assayed. The PS activity (PSAct) was determined by a clotting method using a commercial hit (Staclot Protein S, Diagnostica Stago, Am&es, France).
Key words: Protein S, normal range, sex-dependence, age-dependence. Corresponding autor: Dr Montserrat Borrell. Unitat d’Hemostlia i Trombosi, Hospital de la Santa Creu i Sant Pau. Avgda Sant Antoni MB Claret, 167. 08025 Barcelona. Spain.
149
150
Vol. 73, No. 2
YOUNG WOMEN AND PS NORMAL RANGE
Total PS antigen (tPSAg) was determined using a commercial ELISA Kit (Asserachrom protein S, Diagnostica Stago, Asnieres, France), with the following modifications: Standard and plasma samples were diluted 1:250, and were incubated with the first antibody overnight. Both modifications were performed in order to promote the dissociation of the C,b-binding protein (C,bBP)-PS complex. Free PS antigen (fPSAg) was determined using the same commercial ELISA Kit. In order to precipitate the C,bBP-PS complex, ppp was mixed with polyethylene-glycol6,OOO(PEG-6,000) 3.75 % (7) (300 ,~l ppp with 53 ,~l 25 % w/v solution of PEG-6,000 in an Eppendorf tube), after vigorous vortexing, it was incubated for 30 min in an ice bath and then centrifuged for 2 min at 12,000 gin an Eppendorf centrifuge. Supernatants were then assayed according to the manufacturer’s instructions. Standard curves were drawn from a series of dilutions between 150 - 2.5% of a plasma pool obtained from 40 blood donors.
RESULTS l-
PS values in the four groups and with the three methods showed a wide but normai distribution. For this reason the 5-95 % confidence interval (CI 56) was taken as a normaI range. The mean PS values and 5-95 % CI are expressed in TABLE I. TABLE I
109
155-75
109
160-74
108
14084
102
13980
90
130-74
107
133-82
116
161-80
101
155-63
110
180-71
88
128-60
78
115-52
88
119-64
Comparison between groups was performed using the Student’s t Test (cu= 0.05) and p values are expressed in TABLE II. When comparing the young female group (18-45 years) with the other three groups, we found significantly lower values (p <: 0.05) with the three methods. In contrast, no differences were found by any of the three methods, when comparing younger and older males (p> 0.1). When comparing the female and male groups over 45 years we only found significant differences with the fPSAg method (p = 0.01).
TABLE II PSAct
Groups
p*
fI-Ag
p’
tPSAg
p*
df c 45
0.25
0.30
0.78
9~451
9145
0.02
0.02
< 0.001
Q < 45 1
&
45
c 0.001
< 0.001
< 0.001
Q > 45 I
&-
45
0.24
0.01
0.84
.A
45 I
* p values refered to Student’s t-test.
YOUNG WOMEN AND PS NORMAL
Vol. 73, No. 2
2-
151
RANGE
The relationship beween PSAct and fPSAg studied by a linear model (y = a + bx) showed a correlation coefficient r = 0.77, p< 0.001, using the group of 130 blood donors and the 46 deficient patients. The correlation coefficient improved when we used the multiplicative model (y = axb) r= 0.87, p
PS act %
I
I.,
1
(
1,
..I,.
.
,
.
.:
ow.~“~~
0
I
I
40
1
.
.
1
,
,
2.
‘.
.:
.
.
*
,
-
I
,
120
80
.
.
..-,__--
,
__‘-
_.’
..... _>_-
I
160
,
,
_,,...-‘,_.
,
.
4 % 200
f PS ag
FIG. 1: Regression analysis by a multiplicative model (v = axb) between PSAct and fPSAg using the group of 130 healthy donors and the 46 PS deficient patients. Correlation coefficient was r = 0.87, p < 0.001.
DISCUSSION Results show a lower PS plasma level in women under 46 years as compared to older women and to men irrespective of age. In previous studies (4,6,8) PS levels were shown to display a strong sex-dependence and, although women have lower PS values, no differences have been mentioned related to age. PS levels in females seem to be influenced by their hormonal state (5,9,10) and this argument supports our finding on the differences between women younger than 46 years and older women. Although 46 is commonly accepted as the cut-off point for menopause, it can not be applied for each individual, which might explain the significant differences found for fPSAg between both sexes over 46 years. When comparing both immunological and functional methods for fPSAg assessment, we found a positive correlation r= 0.77 in a linear model and r= 0.87 in a multiplicative model, suggesting that the relationship between the two methods decreases when PS levels increase. Although both methods measure the same plasma compound, the correlation between them is still far from r = 1, suggesting methodological differences. The main aim of this study was to determine normal ranges for functional and antigen PS plasma levels in order to accurately diagnose deficient states. According to our results, we conclude that females under 46 years have a lower PS plasma range. A cut-off at 45 years should therefore be taken for women in order to avoid misestimations of deficient or normal individuals.
Acknowledgements This work was supported in part by a grant from Ministerio de Education y Ciencia (DGICYT PM90-0064).
152
YOUNG WOMEN AND PS NORMAL RANGE
Vol. 73, No. 2
REFERENCES 1.
10.
BERTINA, R.M., VAN WIJNGAARDEN, A, REINALDA-POOT, J., POORT, S.R., BOM, V.J.J. Dertermination of plasma protein S -the protein cofactor of activated protein C. Tbromb Haemostas. $3, 268-272, 1985. ENGESSER, L., BROEKMANS, A.W., BRIET, E., BRUMMER, E.J.P., BERTINA, R.M. Hereditary protein S deficiency: Clinical manifestations. Ann Intern Med. l.& 677-682, 1987. SUZUKI, K., NISHIOKA, J. Plasma protein S activity measured using Protac, a snake venom derivated activator of protein C. Thromb. Res. g, 241-251, 1988. EDSON, J.R., VOGT, J.M., HUESMAN, D.A. Laboratory diagnosis of inherited protein S deficency. Am.J.Clin.Pathol. 94, 2, 176-186, 1990. MALM, J., LAURELL, M., DAHLBACK, B. Changes in the plasma levels of vitamin K-dependent protein C and S and of C4b-binding protein during pregnancy and oral contraception. Br. J. of Haematol. 6& 437-443, 1988. BOERGER, L.M., MORRIS, P.C., THURNAU, G.R., ESMON, C.T., COMP, P.C. Oral contraceptives and gender affect protein S status. Blood. 69, 692-694, 1987. COMP, P.C., DORAY, D., PATTON, D., ESMON, C. An abnormal plasma distribution of protein S occurs in functional protein S deticency. Blood a, 2, 504-508. 1986. CADROY Y., DAVIAUD P., SAMN S., SIB P., BONEU B.: Distribution of 16 hemostatic laboratory variables assayed in 100 blood donors. Nouv.Rev.F_r.Hematol. a, 259-264, 1990. FERNANDEZ, J.A., ESTELLES, A, GILABERT, J., ESPANA, F., AZNAR, J. Functional and immunologic protein S in normal pregnant women and in fug-term newborns. Thromb. Haemostas. tjl, 3, 474-478, 1989. PETERS, M. Acquired Protein S Deficiency Might Be Associated with a Prethrombotic State During Estrogen Treatment for Tail Stature. Thromb. Haemostas. @, 3, 371-372, 1992.
Pergamon
MINI REPORT
THE INFLUENCE OF LOW PROTEIN S PLASMA LEVELS IN YOUNG WOMEN, ON THE DEFINITION OF NORMAL RANGE.
M. Gari, L. Falkon, T. Urrutia, C. VallvC, M. Borrell, J. Fontcuberta. Unitat d’Hemost&ia i Trombosi. Hospital de la Santa Creu i Sant Pau. Barcelona, Spain.
(Received
15 April 1993 by Editor C. Soria; accepted 5 November
1993)
The normal range for plasma Protein S (PS) concentration using different methods has been reported by several investigators, and values show evident discrepancies (1,2,3). The reasons which might al&t these results are still unclear, although some reports show the influence of sex in plasma PS levels (4) and others had reported changes during pregnancy and in women on oral contraceptives (5,6). With the purpose to define the normal range for accurately diagnose deficient states (congenital and/or acquired), we study: l- the possible influence of sex and/or age in plasma PS levels (for three different methods) in a healthy population (n= 130) divided into 4 groups according to age and sex. 2- the relationship between immunological and functional methods for PS quantification.
SUBJECTS. MATERIALS AND METHODS 130 plasma samples were obtained from healthy blood donors and were used to establish the normal range and the relationship between methods. They were distributed into 4 groups: a) 28 males, mean age 53.6 years (range 46 to 65). b) 23 females, mean age 53.8 years (range 46 to 65). c) 38 males, mean age 32.6 years (range 18 to 45). d) 41 females, mean age 35.3 years (range 18 to 45). 46 different samples were obtained from non-anticoagulated individuals with low PS plasma levels, as detected by previous determinations. This group together with the 130 blood donors plasmas were used to study the relationship between methods. Following 12 hours’ fasting, samples were drawn between 9.00 and 11.00 am, by veinpuncture into vacutainer tubes containing 13 mM trisodum citrate. Platelet poor plasma (ppp) was prepared by centrifugation at 3,500 rpm for 20 minutes at room temperature. Plasma samples were stored at -40°C until assayed. The PS activity (PSAct) was determined by a clotting method using a commercial hit (Staclot Protein S, Diagnostica Stago, Am&es, France).
Key words: Protein S, normal range, sex-dependence, age-dependence. Corresponding autor: Dr Montserrat Borrell. Unitat d’Hemostlia i Trombosi, Hospital de la Santa Creu i Sant Pau. Avgda Sant Antoni MB Claret, 167. 08025 Barcelona. Spain.
149
150
Vol. 73, No. 2
YOUNG WOMEN AND PS NORMAL RANGE
Total PS antigen (tPSAg) was determined using a commercial ELISA Kit (Asserachrom protein S, Diagnostica Stago, Asnieres, France), with the following modifications: Standard and plasma samples were diluted 1:250, and were incubated with the first antibody overnight. Both modifications were performed in order to promote the dissociation of the C,b-binding protein (C,bBP)-PS complex. Free PS antigen (fPSAg) was determined using the same commercial ELISA Kit. In order to precipitate the C,bBP-PS complex, ppp was mixed with polyethylene-glycol6,OOO(PEG-6,000) 3.75 % (7) (300 ,~l ppp with 53 ,~l 25 % w/v solution of PEG-6,000 in an Eppendorf tube), after vigorous vortexing, it was incubated for 30 min in an ice bath and then centrifuged for 2 min at 12,000 gin an Eppendorf centrifuge. Supernatants were then assayed according to the manufacturer’s instructions. Standard curves were drawn from a series of dilutions between 150 - 2.5% of a plasma pool obtained from 40 blood donors.
RESULTS l-
PS values in the four groups and with the three methods showed a wide but normai distribution. For this reason the 5-95 % confidence interval (CI 56) was taken as a normaI range. The mean PS values and 5-95 % CI are expressed in TABLE I. TABLE I
109
155-75
109
160-74
108
14084
102
13980
90
130-74
107
133-82
116
161-80
101
155-63
110
180-71
88
128-60
78
115-52
88
119-64
Comparison between groups was performed using the Student’s t Test (cu= 0.05) and p values are expressed in TABLE II. When comparing the young female group (18-45 years) with the other three groups, we found significantly lower values (p <: 0.05) with the three methods. In contrast, no differences were found by any of the three methods, when comparing younger and older males (p> 0.1). When comparing the female and male groups over 45 years we only found significant differences with the fPSAg method (p = 0.01).
TABLE II PSAct
Groups
p*
fI-Ag
p’
tPSAg
p*
df c 45
0.25
0.30
0.78
9~451
9145
0.02
0.02
< 0.001
Q < 45 1
&
45
c 0.001
< 0.001
< 0.001
Q > 45 I
&-
45
0.24
0.01
0.84
.A
45 I
* p values refered to Student’s t-test.
YOUNG WOMEN AND PS NORMAL
Vol. 73, No. 2
2-
151
RANGE
The relationship beween PSAct and fPSAg studied by a linear model (y = a + bx) showed a correlation coefficient r = 0.77, p< 0.001, using the group of 130 blood donors and the 46 deficient patients. The correlation coefficient improved when we used the multiplicative model (y = axb) r= 0.87, p
PS act %
I
I.,
1
(
1,
..I,.
.
,
.
.:
ow.~“~~
0
I
I
40
1
.
.
1
,
,
2.
‘.
.:
.
.
*
,
-
I
,
120
80
.
.
..-,__--
,
__‘-
_.’
..... _>_-
I
160
,
,
_,,...-‘,_.
,
.
4 % 200
f PS ag
FIG. 1: Regression analysis by a multiplicative model (v = axb) between PSAct and fPSAg using the group of 130 healthy donors and the 46 PS deficient patients. Correlation coefficient was r = 0.87, p < 0.001.
DISCUSSION Results show a lower PS plasma level in women under 46 years as compared to older women and to men irrespective of age. In previous studies (4,6,8) PS levels were shown to display a strong sex-dependence and, although women have lower PS values, no differences have been mentioned related to age. PS levels in females seem to be influenced by their hormonal state (5,9,10) and this argument supports our finding on the differences between women younger than 46 years and older women. Although 46 is commonly accepted as the cut-off point for menopause, it can not be applied for each individual, which might explain the significant differences found for fPSAg between both sexes over 46 years. When comparing both immunological and functional methods for fPSAg assessment, we found a positive correlation r= 0.77 in a linear model and r= 0.87 in a multiplicative model, suggesting that the relationship between the two methods decreases when PS levels increase. Although both methods measure the same plasma compound, the correlation between them is still far from r = 1, suggesting methodological differences. The main aim of this study was to determine normal ranges for functional and antigen PS plasma levels in order to accurately diagnose deficient states. According to our results, we conclude that females under 46 years have a lower PS plasma range. A cut-off at 45 years should therefore be taken for women in order to avoid misestimations of deficient or normal individuals.
Acknowledgements This work was supported in part by a grant from Ministerio de Education y Ciencia (DGICYT PM90-0064).
152
YOUNG WOMEN AND PS NORMAL RANGE
Vol. 73, No. 2
REFERENCES 1.
10.
BERTINA, R.M., VAN WIJNGAARDEN, A, REINALDA-POOT, J., POORT, S.R., BOM, V.J.J. Dertermination of plasma protein S -the protein cofactor of activated protein C. Tbromb Haemostas. $3, 268-272, 1985. ENGESSER, L., BROEKMANS, A.W., BRIET, E., BRUMMER, E.J.P., BERTINA, R.M. Hereditary protein S deficiency: Clinical manifestations. Ann Intern Med. l.& 677-682, 1987. SUZUKI, K., NISHIOKA, J. Plasma protein S activity measured using Protac, a snake venom derivated activator of protein C. Thromb. Res. g, 241-251, 1988. EDSON, J.R., VOGT, J.M., HUESMAN, D.A. Laboratory diagnosis of inherited protein S deficency. Am.J.Clin.Pathol. 94, 2, 176-186, 1990. MALM, J., LAURELL, M., DAHLBACK, B. Changes in the plasma levels of vitamin K-dependent protein C and S and of C4b-binding protein during pregnancy and oral contraception. Br. J. of Haematol. 6& 437-443, 1988. BOERGER, L.M., MORRIS, P.C., THURNAU, G.R., ESMON, C.T., COMP, P.C. Oral contraceptives and gender affect protein S status. Blood. 69, 692-694, 1987. COMP, P.C., DORAY, D., PATTON, D., ESMON, C. An abnormal plasma distribution of protein S occurs in functional protein S deticency. Blood a, 2, 504-508. 1986. CADROY Y., DAVIAUD P., SAMN S., SIB P., BONEU B.: Distribution of 16 hemostatic laboratory variables assayed in 100 blood donors. Nouv.Rev.F_r.Hematol. a, 259-264, 1990. FERNANDEZ, J.A., ESTELLES, A, GILABERT, J., ESPANA, F., AZNAR, J. Functional and immunologic protein S in normal pregnant women and in fug-term newborns. Thromb. Haemostas. tjl, 3, 474-478, 1989. PETERS, M. Acquired Protein S Deficiency Might Be Associated with a Prethrombotic State During Estrogen Treatment for Tail Stature. Thromb. Haemostas. @, 3, 371-372, 1992.