Journal of Immunological Methods, 133 (1990) 21-29
21
Elsevier JIM05687
A turbidimetric assay in an ELISA reader for the determination of mononuclear phagocyte procoagulant activity Thomas W. Jungi Institute of Veterinary Virology, University of Berne, Berne, Switzerland (Received 5 January 1990, revised received 20 April 1990, accepted 1 June 1990)
A method is described for the simultaneous determination of plasma recalcification time in 60-96 replicates. It is based on turbidimetry in a thermostatic (37°C) ELISA reader photometer. Dose-reponse curves with thromboplastin showed a double-logarithmic correlation between recalcification time and thromboplastin concentration. Various ways of determining the coagulation time by turbidimetry were compared. Samples of mononuclear cells or monocyte-derived macrophages showed that procoagulant activity (PCA), expressed in thromboplastin units, was directly proportional to the cell number. The method may be of value for the determination of mononuclear phagocyte PCA and for other applications of plasma recalcification time. Key words: Proeoagulant activity; Thromboplastin; Monocyte; Macrophage; Methodology; Coagulation; Endotoxin
Introduction
Monocytes and macrophages participate actively in hemostasis and coagulation in various ways. Upon appropriate stimulation, they express tissue thromboplastin and a variety of other coagulation factors, thereby accelerating coagulation (procoagulant activity, PCA) (reviewed in Edwards and Rickles, 1984; Lyberg, 1984; Herin, 1986; Morgan et al., 1988). They are involved in the removal and degradation of fibrin (Rajagopalan and Pizzo, 1986), and express surface constituents capable of mediating the binding of thrombin-activated, but not resting platelets (Jungi et al., 1986; Silverstein and Nachman, 1987; Larsen et al., 1989). Lipid mediators including platelet activating factor (PAF) and thromboxane pro-
Correspondence to: T.W. Jtmgi, Institute of Veterinary Virology, L~inggass-Str. 122, CH-3012 Bern, Switzerland.
mote platelet aggregation (Altieri and Mannucci, 1986; Braquet and Rola Pleszczynski, 1987), and cytoldnes such as interleukin-1 and tumour necrosis factor-a upregulate procoagulatory and downregulate anticoagulatory proteins on the surface of endothelial cells (Bevilacqua et al., 1985; Nawroth and Stem, 1985). The determination of PCA expressed by mononuclear phagocytes has become an often-used method for determining the functional state of these cells, since several distinct means of cell stimulation lead to enhanced expression of PCA. Appropriate stimuli encompass endotoxin (Shands, 1983), T cell-derived mediators (Geczy, 1984; Gregory et al., 1986; Ryan and Geczy, 1988) and as yet ill-characterized plasma factors (Edwards and Perla, 1984). The level of PCA expression may be representative for mononuclear phagocyte-mediated pathology in a variety of diseases. These include gram-negative septic shock associated with hemorrhagic thrombosis (Lyberg,
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
22 1984; Almdahl and Osterud, 1987), neoplastic disease (Osterud and Due, 1984; Dasmahapatra et al., 1987; Morgan et al., 1988), surgical intervention (Osterud and Due, 1984) and viral infection (Dindzans et al., 1985; Peterhans et al., 1988). It may also serve as a hallmark for mononuclear phagocyte activation or a state of cell-mediated immunity (Hopper et al., 1981; Gregory et al., 1986; Moon and Geczy, 1988). Conventionally, PCA is measured by determining the acceleration in blood clotting after the addition of Ca 2÷ to citrated plasma supplemented with mononuclear phagocytes (or their secretion products). Various types of coagulometers render this test more quantitative than the classical procedure involving hook and stop-watch. Here we describe a microtiter plate-based method which permits the simultaneous determination of clotting times in 60-96 samples using a thermostatic ELISA reader photometer. This simplified procedure for the measurement of PCA may be used as a screening test for determining mononuclear phagocyte function in disease processes or under experimental conditions and for determining the degree of endotoxin contamination.
Materials and methods
Reagents Plasma from 20 volunteers (50 ml/donor) was pooled, rendered platelet-free by centrifugation (20 min at 2000 × g), distributed into aliquots and stored frozen at - 8 0 ° C. Thromboplastin in the form of lyophilized human placenta (Thromborel S, TS) was purchased from Behringwerke, Marburg, F.R.G. Doubly acid-washed diatomaceous earth ('celite') was obtained from Sigma (St. Louis, MO). Cell culture media and Ficoll-Hypaque was from Seromed (Munich, F.R.G.). Inorganic compounds were purchased from Merck (Darmstadt, F.R.G.). Cell isolation and culture Mononuclear cells were isolated from fresh venous blood anticoagulated with ACD by a modified Ficoll-Hypaque procedure, aimed at minimizing platelet contamination and exposure of the
cells to endotoxin (Jungi et al., 1986). For some experiments, monocytes purified as described by selective adherence and detachment were allowed to differentiate into macrophages, using hydrophobic teflon culture bags (Jungi and Hafner, 1986; Jungi et al., 1986).
ELISA reader photometers and software for converting recalcification times into PCA units Several ELISA readers permitting kinetic photometry were used. For example, an MR600 (Dynatech, Embrach, Switzerland) equipped with a water-perfused thermostatic plate-holder was used in combination with the 'Immunosoft' program and an AT-compatible personal computer. However, this software was found to be unsatisfactory for the desired purpose and more appropriate software is now under development. Two other readers, the Anthos Labtec 2001 (Kontron Zurich, Switzerland) and the SLT EAR-340 (Tecan, Hombrechtikon, Switzerland) were operated in a 37°C room. Both of these readers had standard software capable of handling kinetic data, but adaptations using a standard curve had to be done off-line by log-linear regression analysis. Thermostatic readers from Anthos, SLT and Flow Laboratories (Mecklenburg b. Bonn, F.R.G.) have subsequently become available and software permitting the direct conversion of recalcification times into PCA units is being developed. In most of the present assays, however, the Thermomax reader (Molecular Devices Co., Palo Alto, CA) in combination with the 'Softmax' software provided by the manufacturer and an IBM AT-compatible personal computer were used. The present version of the Softmax program (2.01) was satisfactory for the desired purpose since it allowed direct conversion of recalcification times (defined by the time until Omax(maximal increment in OD between two measurements) was reached) into PCA units, using a standard curve of various thromboplastin concentrations. The calculation of means, standard deviations, and coefficients of variation for a set of replicates was possible with this software. Those readers performing the measurements with little or no agitation of the plate were preferred. An advantage of the Thermomax reader was that the plate could be measured with the lid on, thereby
23 minimizing fluid loss due to ventilation. Furthermore, temperature control was more precise when evaporation was minimized. A condensate forruing on the inner surface of the lid was completely prevented by preparing the lid with an anti-foam fluid provided by the reader manufacturer.
Turbidimetric procoagulant actioity test Mononuclear cells were washed, resuspended in physiologic saline and adjusted to the desired monocyte concentration. In some experiments, cells kept at 4 ° C overnight, or kept frozen ( - 2 0 ° C ) and lysed by repeated freeze-thawing were used. An irradiation-sterilized flat-bottom 96-well microtiter plate (Nunc, Roskilde, Denmark) was placed in a waterbath containing a custom-made thermoblock for microtiter plates. This block was perfused by and immersed in water which permitted the adjustment of the well contents to 37°C + 0.2°C in the inner 60 wells of the plate. If edge and corner wells were included in the assay, a slightly larger variation had to be taken into account. Each well received 100 #1 of fresh-frozen pooled citrated human plasma. After a sufficient time to allow for temperature adjustments (at least 20 min), 100/~1 of a cell suspension containing either intact or lysed cells or cell culture supernatants, were added. Some wells received 100/~1 of a TS dilution in a range between 102-106-fold. After temperature adjustment, plates and a CaC12 solution (25 mM) prewarmed at 37°C were transferred to the ELISA reader. At time 0, warm CaC12 was dispensed with a multichannel pipette (100 #l/well), immediately prior to the onset of the kinetic measurement of OD under thermostatic (37 ° C) conditions. The well contents were mixed by the jet of CaC12 pipetted into the wells, without any need for further agitation. Total reading time varied between 20 and 30 rain, the intervals between individual measurements lying between 10 and 30 s, depending on the data storage capacity of the reader software. Measurements were routinely taken at a wavelength of 365 n.m, but measurement within the visible range was possible without any loss in precision.
R ~
Optimal assay conditions for turbidimetric measurement of plasma coagulation Preliminary experiments were aimed at deterrnining the influence of Ca 2+ concentration, medium composition, agitation, geometry of the microtiter plates and wavelength of measurement, on the turbidimetric plasma coagulation curve. A final Ca 2÷ concentration of 8-10 mM was found to be optimal, thus confirming the results of others (Shands, 1983; Gregory et al., 1986). In order to prevent salt precipitation, cells to be assayed have to be resuspended in saline or a culture medium low in phosphate. Tests with various plasma concentrations showed that 33% plasma was optimal. both flat-bottom and round-bottom microtiter plates could be used. Using a shorter wave length, the difference in OD between uncoagulated and coagulated plasma was larger. Various methods of agitating the well contents suggested that the coagulation time, in absolute terms, was dependent on the intensity of initial agitation, particularly when the Ca 2÷ concentration was low. In order to keep the interval between Ca 2÷ addition and onset of measurement short, well contents were mixed by the jet of the multipipette filled with CaC12. Using 8.3 mM final Ca 2÷ concentration, half-maximal coagulation, as defined by half-maximal OD, was reached within 600-1000 s, depending on the storage time of the plasma. The influence of added thromboplastin on the turbidimetric coagulation curve Using the assay conditions described in the materials and methods section without cells, various TS concentrations were added to plasma-containing wells, and coagulation was initiated by the addition of Ca 2+. Plasma coagulation was accelerated by TS in a dose-dependent-fashion and the resulting turbidimetric curves became steeper with higher TS concentrations (Fig. 1). Since both the initial OD and the final OD differed between wells, depending on the TS concentration (Fig. 1), various criteria for defining half-maximal coagulation were considered. For example, the latter could be defined as the median OD between starting
24
5
Time (rain) 10
15
20
1.6 1.4
~ 1.2 r,3 5oo
o
1.0
0.8
/I,'////I,,W 2o i -
0.6
o i 2 3 Thromboplastin (Ioglo
* units)
Fig. 1. Temporal traces of O D increments of plasma containing various amounts of thromboplastin (Thromborel S) and recalcified with 8.3 m M Ca 2+ at time 0. Figures refer to lOgl0 TS units, 1 U being a 106-fold dilution of TS. Curves within (and including) dashed lines correspond to the recommended working range. Insert: half-maximal d o t t i n g time (time until Vmax is reached) as a function of thromboplastin concentration. Solid line: calculated linear regression line within the recomended working range (r = 0.986). Dashed line: calculated regression of 2nd order over whole test range (r = 0.998).
OD and final OD. This median could be determined either for each well individually, or one median for the whole plate could be calculated. Alternatively, half-maximal coagulation could be defined as the time when Vmax was reached. The latter could be calculated from linear regression analyses, involving 2 or more points. Fig. 2 shows that within the range of TS diluted 102-105-fold, the method of calculating the median OD for a whole microtiter plate and determining the time until median OD was reached, provided the best correlation in a double-logarithmic plot, although the other methods of calculating half-maximal coagulation were also found to be acceptable (Figs. 1 and 2). A method of eliminating differences in OD at the beginning of the assay was to register OD differences between two measurements, instead of absolute OD values, as provided by the 'Softmax' software of the Thermomax reader. Using this commercial standard software, it is possible to determine the time elapsed before the maximal slope between any number of points is reached. Chosing an interval of 10 s and 3 points for regression analysis, data such as those of Fig. 1 (insert) are obtained. Thus, the ELISA readerbased turbidimetric recalcification time assay
shows the expected inverse proportionality between thromboplastin concentration and coagulation time, although the recalcification times obtained differ from those derived using conventional methodology. Using an 8- or 12-channel multipipette, it was not possible to load all wells simultaneously with Ca 2÷, and this represented an inprecision when taking the time of the first OD determination as time 0. This systematic error was significant for short recalcification times ( < 100 s), but negligible for longer times. Using the Softmax program, we did not compensate for these time delays; the recommended working range was therefore between 1 and 103 TS units. When using a device allowing simultaneous pipetting for 96 wells, or with software allowing time 0 compensation, the working range could be extended to five decades. In order to eliminate the contribution of the intrinsic coagulation pathway, plasma was pretreated with diatomaceous earth, thereby absorbing Hageman factor/factor XII of the intrinsic pathway. In Fig. 3, coagulation times with or without intrinsic pathway blockade are compared. The intrinsic pathway had little influence on coagulation time at high TS concentrations, but contributed significantly to coagulation at low TS concentrations. Intrinsic pathway blockade was therefore a means of rendering PCA determinations more sensitive.
Determination of mononuclear phagocyte procoagulant activity Monocytes were cultured for 20 h in the presence or absence of LPS, washed and then added in various numbers to microtiter plates containing citrated plasma. After temperature adjustment, Ca 2+ was added, and the increase in OD determined turbidimetrically. The half-maximal coagulation time obtained was converted into TS units, one unit being the activity of a 106-fold dilution of TS. The magnitute of PCA depended on the monocyte pretreatment and on the cell number (Fig. 4, left-hand panel). Supernatants of mononuclear cell cultures contained much lower amounts of PCA (Fig. 4, right-hand panel). Monocyte-derived macrophages, which had been primed with interferon-y or left unprimed, were also exposed to LPS, and PCA was determined 1 day
25
1000 ..
Median O.D. (plate average) (r = 0 . 9 9 9 )
Median O.D. for each well •. (r = 0.99)
0,,
O't-e: O 100
O... 0. &
O'-O.
0,~
"U tO 0
"~" 1000 E
• .
Maximal slope (2 points) (r = 0.99)
-.
°'e.e
Maximal slope (4 points) (r = 0.99)
ILII "O. O
"O k
100
,\
O,
I%
o,.
O.
O.
1o
,
i
10 5
i
i
10 3
i
i
10
10 5
i
i
I
103
i
10
Thromboplastin dilution
Fig. 2. Correlation between thromboplastin (Thromborel S) dilution and coagulation time. The latter was determined in four differvnt ways (see text)• Dotted lines: linear regression lines.
later. Again, a cell dose-dependent and pretreatment-dependent expression of PCA was noted, whereas macrophage supernatants were found to 1000 : ~
600
?
5OO
with celite
"
21
Elsevier JIM05687
A turbidimetric assay in an ELISA reader for the determination of mononuclear phagocyte procoagulant activity Thomas W. Jungi Institute of Veterinary Virology, University of Berne, Berne, Switzerland (Received 5 January 1990, revised received 20 April 1990, accepted 1 June 1990)
A method is described for the simultaneous determination of plasma recalcification time in 60-96 replicates. It is based on turbidimetry in a thermostatic (37°C) ELISA reader photometer. Dose-reponse curves with thromboplastin showed a double-logarithmic correlation between recalcification time and thromboplastin concentration. Various ways of determining the coagulation time by turbidimetry were compared. Samples of mononuclear cells or monocyte-derived macrophages showed that procoagulant activity (PCA), expressed in thromboplastin units, was directly proportional to the cell number. The method may be of value for the determination of mononuclear phagocyte PCA and for other applications of plasma recalcification time. Key words: Proeoagulant activity; Thromboplastin; Monocyte; Macrophage; Methodology; Coagulation; Endotoxin
Introduction
Monocytes and macrophages participate actively in hemostasis and coagulation in various ways. Upon appropriate stimulation, they express tissue thromboplastin and a variety of other coagulation factors, thereby accelerating coagulation (procoagulant activity, PCA) (reviewed in Edwards and Rickles, 1984; Lyberg, 1984; Herin, 1986; Morgan et al., 1988). They are involved in the removal and degradation of fibrin (Rajagopalan and Pizzo, 1986), and express surface constituents capable of mediating the binding of thrombin-activated, but not resting platelets (Jungi et al., 1986; Silverstein and Nachman, 1987; Larsen et al., 1989). Lipid mediators including platelet activating factor (PAF) and thromboxane pro-
Correspondence to: T.W. Jtmgi, Institute of Veterinary Virology, L~inggass-Str. 122, CH-3012 Bern, Switzerland.
mote platelet aggregation (Altieri and Mannucci, 1986; Braquet and Rola Pleszczynski, 1987), and cytoldnes such as interleukin-1 and tumour necrosis factor-a upregulate procoagulatory and downregulate anticoagulatory proteins on the surface of endothelial cells (Bevilacqua et al., 1985; Nawroth and Stem, 1985). The determination of PCA expressed by mononuclear phagocytes has become an often-used method for determining the functional state of these cells, since several distinct means of cell stimulation lead to enhanced expression of PCA. Appropriate stimuli encompass endotoxin (Shands, 1983), T cell-derived mediators (Geczy, 1984; Gregory et al., 1986; Ryan and Geczy, 1988) and as yet ill-characterized plasma factors (Edwards and Perla, 1984). The level of PCA expression may be representative for mononuclear phagocyte-mediated pathology in a variety of diseases. These include gram-negative septic shock associated with hemorrhagic thrombosis (Lyberg,
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
22 1984; Almdahl and Osterud, 1987), neoplastic disease (Osterud and Due, 1984; Dasmahapatra et al., 1987; Morgan et al., 1988), surgical intervention (Osterud and Due, 1984) and viral infection (Dindzans et al., 1985; Peterhans et al., 1988). It may also serve as a hallmark for mononuclear phagocyte activation or a state of cell-mediated immunity (Hopper et al., 1981; Gregory et al., 1986; Moon and Geczy, 1988). Conventionally, PCA is measured by determining the acceleration in blood clotting after the addition of Ca 2÷ to citrated plasma supplemented with mononuclear phagocytes (or their secretion products). Various types of coagulometers render this test more quantitative than the classical procedure involving hook and stop-watch. Here we describe a microtiter plate-based method which permits the simultaneous determination of clotting times in 60-96 samples using a thermostatic ELISA reader photometer. This simplified procedure for the measurement of PCA may be used as a screening test for determining mononuclear phagocyte function in disease processes or under experimental conditions and for determining the degree of endotoxin contamination.
Materials and methods
Reagents Plasma from 20 volunteers (50 ml/donor) was pooled, rendered platelet-free by centrifugation (20 min at 2000 × g), distributed into aliquots and stored frozen at - 8 0 ° C. Thromboplastin in the form of lyophilized human placenta (Thromborel S, TS) was purchased from Behringwerke, Marburg, F.R.G. Doubly acid-washed diatomaceous earth ('celite') was obtained from Sigma (St. Louis, MO). Cell culture media and Ficoll-Hypaque was from Seromed (Munich, F.R.G.). Inorganic compounds were purchased from Merck (Darmstadt, F.R.G.). Cell isolation and culture Mononuclear cells were isolated from fresh venous blood anticoagulated with ACD by a modified Ficoll-Hypaque procedure, aimed at minimizing platelet contamination and exposure of the
cells to endotoxin (Jungi et al., 1986). For some experiments, monocytes purified as described by selective adherence and detachment were allowed to differentiate into macrophages, using hydrophobic teflon culture bags (Jungi and Hafner, 1986; Jungi et al., 1986).
ELISA reader photometers and software for converting recalcification times into PCA units Several ELISA readers permitting kinetic photometry were used. For example, an MR600 (Dynatech, Embrach, Switzerland) equipped with a water-perfused thermostatic plate-holder was used in combination with the 'Immunosoft' program and an AT-compatible personal computer. However, this software was found to be unsatisfactory for the desired purpose and more appropriate software is now under development. Two other readers, the Anthos Labtec 2001 (Kontron Zurich, Switzerland) and the SLT EAR-340 (Tecan, Hombrechtikon, Switzerland) were operated in a 37°C room. Both of these readers had standard software capable of handling kinetic data, but adaptations using a standard curve had to be done off-line by log-linear regression analysis. Thermostatic readers from Anthos, SLT and Flow Laboratories (Mecklenburg b. Bonn, F.R.G.) have subsequently become available and software permitting the direct conversion of recalcification times into PCA units is being developed. In most of the present assays, however, the Thermomax reader (Molecular Devices Co., Palo Alto, CA) in combination with the 'Softmax' software provided by the manufacturer and an IBM AT-compatible personal computer were used. The present version of the Softmax program (2.01) was satisfactory for the desired purpose since it allowed direct conversion of recalcification times (defined by the time until Omax(maximal increment in OD between two measurements) was reached) into PCA units, using a standard curve of various thromboplastin concentrations. The calculation of means, standard deviations, and coefficients of variation for a set of replicates was possible with this software. Those readers performing the measurements with little or no agitation of the plate were preferred. An advantage of the Thermomax reader was that the plate could be measured with the lid on, thereby
23 minimizing fluid loss due to ventilation. Furthermore, temperature control was more precise when evaporation was minimized. A condensate forruing on the inner surface of the lid was completely prevented by preparing the lid with an anti-foam fluid provided by the reader manufacturer.
Turbidimetric procoagulant actioity test Mononuclear cells were washed, resuspended in physiologic saline and adjusted to the desired monocyte concentration. In some experiments, cells kept at 4 ° C overnight, or kept frozen ( - 2 0 ° C ) and lysed by repeated freeze-thawing were used. An irradiation-sterilized flat-bottom 96-well microtiter plate (Nunc, Roskilde, Denmark) was placed in a waterbath containing a custom-made thermoblock for microtiter plates. This block was perfused by and immersed in water which permitted the adjustment of the well contents to 37°C + 0.2°C in the inner 60 wells of the plate. If edge and corner wells were included in the assay, a slightly larger variation had to be taken into account. Each well received 100 #1 of fresh-frozen pooled citrated human plasma. After a sufficient time to allow for temperature adjustments (at least 20 min), 100/~1 of a cell suspension containing either intact or lysed cells or cell culture supernatants, were added. Some wells received 100/~1 of a TS dilution in a range between 102-106-fold. After temperature adjustment, plates and a CaC12 solution (25 mM) prewarmed at 37°C were transferred to the ELISA reader. At time 0, warm CaC12 was dispensed with a multichannel pipette (100 #l/well), immediately prior to the onset of the kinetic measurement of OD under thermostatic (37 ° C) conditions. The well contents were mixed by the jet of CaC12 pipetted into the wells, without any need for further agitation. Total reading time varied between 20 and 30 rain, the intervals between individual measurements lying between 10 and 30 s, depending on the data storage capacity of the reader software. Measurements were routinely taken at a wavelength of 365 n.m, but measurement within the visible range was possible without any loss in precision.
R ~
Optimal assay conditions for turbidimetric measurement of plasma coagulation Preliminary experiments were aimed at deterrnining the influence of Ca 2+ concentration, medium composition, agitation, geometry of the microtiter plates and wavelength of measurement, on the turbidimetric plasma coagulation curve. A final Ca 2÷ concentration of 8-10 mM was found to be optimal, thus confirming the results of others (Shands, 1983; Gregory et al., 1986). In order to prevent salt precipitation, cells to be assayed have to be resuspended in saline or a culture medium low in phosphate. Tests with various plasma concentrations showed that 33% plasma was optimal. both flat-bottom and round-bottom microtiter plates could be used. Using a shorter wave length, the difference in OD between uncoagulated and coagulated plasma was larger. Various methods of agitating the well contents suggested that the coagulation time, in absolute terms, was dependent on the intensity of initial agitation, particularly when the Ca 2÷ concentration was low. In order to keep the interval between Ca 2÷ addition and onset of measurement short, well contents were mixed by the jet of the multipipette filled with CaC12. Using 8.3 mM final Ca 2÷ concentration, half-maximal coagulation, as defined by half-maximal OD, was reached within 600-1000 s, depending on the storage time of the plasma. The influence of added thromboplastin on the turbidimetric coagulation curve Using the assay conditions described in the materials and methods section without cells, various TS concentrations were added to plasma-containing wells, and coagulation was initiated by the addition of Ca 2+. Plasma coagulation was accelerated by TS in a dose-dependent-fashion and the resulting turbidimetric curves became steeper with higher TS concentrations (Fig. 1). Since both the initial OD and the final OD differed between wells, depending on the TS concentration (Fig. 1), various criteria for defining half-maximal coagulation were considered. For example, the latter could be defined as the median OD between starting
24
5
Time (rain) 10
15
20
1.6 1.4
~ 1.2 r,3 5oo
o
1.0
0.8
/I,'////I,,W 2o i -
0.6
o i 2 3 Thromboplastin (Ioglo
* units)
Fig. 1. Temporal traces of O D increments of plasma containing various amounts of thromboplastin (Thromborel S) and recalcified with 8.3 m M Ca 2+ at time 0. Figures refer to lOgl0 TS units, 1 U being a 106-fold dilution of TS. Curves within (and including) dashed lines correspond to the recommended working range. Insert: half-maximal d o t t i n g time (time until Vmax is reached) as a function of thromboplastin concentration. Solid line: calculated linear regression line within the recomended working range (r = 0.986). Dashed line: calculated regression of 2nd order over whole test range (r = 0.998).
OD and final OD. This median could be determined either for each well individually, or one median for the whole plate could be calculated. Alternatively, half-maximal coagulation could be defined as the time when Vmax was reached. The latter could be calculated from linear regression analyses, involving 2 or more points. Fig. 2 shows that within the range of TS diluted 102-105-fold, the method of calculating the median OD for a whole microtiter plate and determining the time until median OD was reached, provided the best correlation in a double-logarithmic plot, although the other methods of calculating half-maximal coagulation were also found to be acceptable (Figs. 1 and 2). A method of eliminating differences in OD at the beginning of the assay was to register OD differences between two measurements, instead of absolute OD values, as provided by the 'Softmax' software of the Thermomax reader. Using this commercial standard software, it is possible to determine the time elapsed before the maximal slope between any number of points is reached. Chosing an interval of 10 s and 3 points for regression analysis, data such as those of Fig. 1 (insert) are obtained. Thus, the ELISA readerbased turbidimetric recalcification time assay
shows the expected inverse proportionality between thromboplastin concentration and coagulation time, although the recalcification times obtained differ from those derived using conventional methodology. Using an 8- or 12-channel multipipette, it was not possible to load all wells simultaneously with Ca 2÷, and this represented an inprecision when taking the time of the first OD determination as time 0. This systematic error was significant for short recalcification times ( < 100 s), but negligible for longer times. Using the Softmax program, we did not compensate for these time delays; the recommended working range was therefore between 1 and 103 TS units. When using a device allowing simultaneous pipetting for 96 wells, or with software allowing time 0 compensation, the working range could be extended to five decades. In order to eliminate the contribution of the intrinsic coagulation pathway, plasma was pretreated with diatomaceous earth, thereby absorbing Hageman factor/factor XII of the intrinsic pathway. In Fig. 3, coagulation times with or without intrinsic pathway blockade are compared. The intrinsic pathway had little influence on coagulation time at high TS concentrations, but contributed significantly to coagulation at low TS concentrations. Intrinsic pathway blockade was therefore a means of rendering PCA determinations more sensitive.
Determination of mononuclear phagocyte procoagulant activity Monocytes were cultured for 20 h in the presence or absence of LPS, washed and then added in various numbers to microtiter plates containing citrated plasma. After temperature adjustment, Ca 2+ was added, and the increase in OD determined turbidimetrically. The half-maximal coagulation time obtained was converted into TS units, one unit being the activity of a 106-fold dilution of TS. The magnitute of PCA depended on the monocyte pretreatment and on the cell number (Fig. 4, left-hand panel). Supernatants of mononuclear cell cultures contained much lower amounts of PCA (Fig. 4, right-hand panel). Monocyte-derived macrophages, which had been primed with interferon-y or left unprimed, were also exposed to LPS, and PCA was determined 1 day
25
1000 ..
Median O.D. (plate average) (r = 0 . 9 9 9 )
Median O.D. for each well •. (r = 0.99)
0,,
O't-e: O 100
O... 0. &
O'-O.
0,~
"U tO 0
"~" 1000 E
• .
Maximal slope (2 points) (r = 0.99)
-.
°'e.e
Maximal slope (4 points) (r = 0.99)
ILII "O. O
"O k
100
,\
O,
I%
o,.
O.
O.
1o
,
i
10 5
i
i
10 3
i
i
10
10 5
i
i
I
103
i
10
Thromboplastin dilution
Fig. 2. Correlation between thromboplastin (Thromborel S) dilution and coagulation time. The latter was determined in four differvnt ways (see text)• Dotted lines: linear regression lines.
later. Again, a cell dose-dependent and pretreatment-dependent expression of PCA was noted, whereas macrophage supernatants were found to 1000 : ~
600
?
5OO
with celite
"
