420
PLATELET
RECEPTORS"
ASSAYS AND PURIFICATION
[36]
[36] E v a l u a t i o n o f P l a t e l e t S u r f a c e A n t i g e n s b y F l u o r e s c e n c e Flow Cytometry B y B U R T A D E L M A N , PATRICIA
CARLSON,
a n d ROBERT I. H A N D I N
Introduction Fluorescence flow cytometry has proven to be a useful technique for identifying cell surface-associated antigens. Large numbers of cells can be examined simultaneously and more than one property of each cell analyzed, thereby facilitating the identification of subpopulations. Both intrinsic constitutive membrane proteins and molecules adsorbed or bound to the cell surface can be identified. For example, by using antibodies directed against unique surface markers this method has been used to identify T lymphocyte subtypes within heterogeneous leukocyte preparations. 1 This chapter will focus on methods for the analysis of platelets by fluorescence flow cytometry. As an example, we describe the identification of glycoprotein Ib (GPIb), the platelet receptor for von Willebrand factor (vWF),2 by incubation of platelets with anti-GPIb antibody. Other investigators have used similar methods to study the glycoprotein IIb-IIIa complex 3 and platelet-associated immunoglobulin, 4-6 complement, 7 blood group antigens,8 and decay-accelerating factor. 9 The technique of fluorescence flow cytometry can also be used to study the binding of proteins like vWF, fibrinogen, and fibronectin to the platelet surface. In addition, the method can be used to screen monoclonal and polyclonal antibodies directed against platelet antigens. For a complete discussion of the theory and instrument design underlying the development of fluorescence flow cytometry equipment and
I E. L. Reinherz and S. F. Schlossman, Cell (Cambridge, Mass.) 19, 821 (1980). 2 B. Adelman, A. D. Michelson, R. I. Handin, and K. A. Ault, Blood 66, 423 (1985). 3 L. K. Jennings, R. A. Ashmun, W. C. Wang, and M. E. Dockter, Blood 68, 173 (1986). 4 j. Lazarchick and S. A. Hall, J. Immunol. Methods 87, 257 (1986). 5 j. Lazarchick, P. V. Genco, S. A. Hall, A. D. Ponzio, and N. M. Burdash, Diagn. lmmunol. 2, 238 (1984). 6 C. S. Rosenfeld and D. C. Bodensteiner, Am. J. Clin. Pathol. 85, 207 (1986). 7 V. Martin, K. A. Ault, and R. I. Handin, Blood 54, Suppl. 1, 112 (1979). 8 R. A. Dunstan and M. B. Simpson, Br. J. Haematol. 61, 603 (1985). 9 A. Nicholson-Weller, J. P. March, C. E. Rosen, D. S. Spicer, and K. F. Austen, Blood 65, 1237 (1985).
METHODS IN ENZYMOLOGY, VOL. 215
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
[36]
PLATELET ANALYSIS BY FLOW CYTOMETRY
421
TABLE I PREPARATION OF PLATELETS FOR ANALYSIS BY FLUORESCENCE FLOW CYTOMETRY
1. 2. 3. 4. 5. 6. 7. 8.
Prepare platelet-rich plasma Fix platelets in 2% (v/v) formaldehyde Wash fixed platelets Incubate platelets with primary or control antibody Wash platelets free of excess antibody Incubate platelets with fluorescein-labeled second antibody Wash platelets free of excess antibody Analyze by flow cytometry
m e t h o d s the reader is referred to a review l° and two texts 11'12 on this subject. Preparation of Platelets and Fluorescent Staining We routinely treat platelets with formaldehyde 13 prior to incubation with antibody (see Table I). A major advantage of formaldehyde fixation is inhibition of degrading e n z y m e s present within platelets that might r e m o v e surface antigens. This is a particular problem with G P I b , as it is rapidly released f r o m the platelet surface by a calcium-dependent protease. ~4Fixation also prevents platelet aggregation during washing steps and enhances the stability of the stained cells. We find that formaldehydetreated platelets can be stored at 4 ° for up to 5 days prior to analysis with minimal loss of the fluorescent signal. The effect of fixation on the immunoreactivity, platelet content, and distribution of any antigen studied must be determined b y the investigator. If fixation alters the i m m u n o r e a c tivity of a particular antigen it is possible to treat platelets with formaldehyde after immunostaining. 8 Whole blood is drawn through a 21-gauge butterfly-type needle into a plastic syringe containing 3.8% (w/v) sodium citrate. Nine parts of blood are mixed with one part anticoagulant using a double-syringe technique. First draw 3 ml of blood into a syringe that is then discarded. A second syringe containing sodium citrate is used to draw the blood. The anticoagu10D. R. Parks and L. A. Herzenberg, this series, Vol. 108, p. 197. 11M. A. Van Dilla, P. N. Dean, O. D. Laerum, and M. R. Melamed, "Flow Cytometry: Instrumentation and Data Analysis." Academic Press, London, 1985. 12H. M. Shapiro, "'Practical Flow Cytometry." Alan R. Liss, New York, 1985. 13j. p. Allain, H. A. Cooper, R. H. Wagner, and K. M. Brinkhous, J. Lab. Clin. Med. 85, 318 (1975). 14D. R. Phillips and M. Jakfibovfi, J. Biol. Chem. 253, 3435 (1978).
422
P L A T E L E T RECEPTORS: ASSAYS A N D P U R I F I C A T I O N
[36]
lated blood is mixed and then centrifuged in polypropylene test tubes at 200 g for 10 min at room temperature. If blood is obtained from a normal donor, 15-30 ml will provide an adequate number of platelets for study although studies have been performed on as little as 1 ml of whole blood. The upper two-thirds of the platelet-rich plasma (PRP) is collected and mixed with an equal volume of buffered 2% (v/v) formaldehyde that has been warmed to 37°. Buffered formaldehyde (2% formaldehyde in TBS: 10 mM Tris, 0.01 M NaC1, pH 7.4) is kept refrigerated and made fresh every week from a stock solution of 37% formaldehyde (Fisher Scientific Co., Fair Lawn, N J). After incubation for 30 min at 37° the platelets are centrifuged at 3200 g for 10 min at 4°, resuspended in washing buffer (TBS containing 2 mM EDTA), and washed twice by centrifugation at 3200 g for 10 min at 4°. After washing, the platelets are suspended in TBS without EDTA. If staining and analysis are not done immediately the platelets should be stored at 4° in TBS containing 0.02% sodium azide. Prior to use, previously stored platelets should be centrifuged and resuspended in fresh buffer. Although we have used TBS in this procedure other physiologic buffers should be equally effective.
Immunofluorescent Staining Glycoprotein Ib was detected on formaldehyde-fixed platelets by indirect immunofluorescent staining (Fig. 1). In our studies we have used both monoclonal and polyclonal antibodies directed against the a chain of GPIb. Monoclonal antibodies were produced by immunizing mice with intact platelets or with purified glycocalicin, a proteolytic fragment of the a chain of GPIb (see [25] in this volume). Polyclonal antibodies were produced by immunizing rabbits with glycocalicin. The second agent is always a fluorescein isothiocyanate (FITC)-conjugated, affinity-purified, F(ab')2 fragment of appropriate specificity (Cooper Biomedical, Inc., Malvern, PA). Formaldehyde-fixed platelets in TBS were diluted to a concentration of 50,000 platelets//.d. They were then incubated for 30 min at 4° with primary antibody (antibody may be in purified form or used as ascites or serum). After incubation the platelets were washed three times by centrifugation (3200 g for 15 min at 4 °) and resuspended in TBS at the original volume. The platelets are then incubated with the appropriate FITC-labeled second antibody for 30 min at 4° and then again washed three times as mentioned above. Each antibody should be used at a saturating concentration, which must be determined by trial and error.
[36]
PLATELET ANALYSIS BY FLOW CYTOMETRY
423
,,,°::200]
03
D IO0-
CONTROL ANTIBODY
Z _1 _.1 W (.)
0
5OO 10~30 FLUORESCENCEINTENSITY
FIG. 1. Fluorescence flow cytometry analysis of GPlb on formaldehyde-treated platelets. Platelets were prepared as described in text and stained with 3G6, a monoclonal antibody directed against the a chain of GPIb or with a control, nonspecific antibody. Fluorescence analysis was performed on an Ortho 50H flow cytometer equipped with a 100-mWargon ion laser operating at 488 nm and 50 mW. Fluorescence intensity is displayed on a linear scale in which the scale units are channel numbers. Each curve represents 10,000 platelets. Both the mean and peak fluorescence signal from the specifically stained platelets is greater than that of the nonspecifically stained platelets.
Fluorescence Flow C y t o m e t r y All flow c y t o m e t e r s currently marketed utilize similar technology for sample presentation, illumination, and raw data capture. Differences exist in the number, power, and placement of light sources and in capabilities for data analysis and presentation. The methods described here should be applicable to machines with even the most basic configuration. Some flow cytometers use a mercury arc lamp rather than a laser as the source of illumination. We have not had adequate experience with such machines to c o m m e n t on their use, so that this discussion is based on experience only with c y t o m e t e r s equipped with a laser. Identification of specific cell populations within a sample prior to fluorescence analysis is usually determined by light scatter measurements. Forward-angle light scatter is proportional to overall cell size, while 90 ° scatter is related to internal cell structure. The two scatter signals taken together can be used to distinguish different cell types in mixed samples (such as whole blood), and to identify dead cells in homogeneous cell preparations. Once a cell population or subpopulation is identified by
424
PLATELET RECEPTORS" ASSAYS AND PURIFICATION
2OO"
[36]
gore I
n~ W m
2-~m B E A D S
:D IOO
Z
.-I _.1 W t_)
360
680
FORWARD SCATTER
FIG. 2. Determination of the appropriate sizing gate for analysis of platelets by flow cytometry. This composite picture includes the forward-anglescatter analysis of 2-p.mbeads and formaldehyde-treated resting platelets. The gate is set to include all particles falling within the region indicated. This region will include approximately80% of the resting platelets that are 2/zm and greater in diameter. Each curve represents 10,000platelets and is displayed on a linear scale in which the scatter units are channel numbers.
its light scatter signal (forward scatter alone or forward- and right-angle scatter), a gate can be drawn that will segregate the population of interest and the machine instructed to collect fluorescent signals from all particles whose scatter characteristics fall within the defined gate. Gate boundaries are delimited by user-defined upper and lower channels. Light scatter from nonspherical cells, such as platelets, is affected by the orientation of each target cell flowing past the laser beam. Nonspherical cells tend to orient with their long axes parallel to the direction of flow. E v e n aligned in this manner, platelets will pass through the laser beam in various orientations and produce a range of scatter signals because of their discoid shape. In addition, aggregated platelets will produce varying scatter signals depending on aggregate size and orientation, We use forward light scatter measurements derived from 2-/xm beads (Polysciences, Warrington, PA) and formaldehyde-treated resting platelets to develop a sizing gate prior to analysis of fluorescent labeled platelets (Fig. 2). We have not found that concurrent 90 ° scatter adds significantly to this process when analyzing a homogeneous sample derived from platelet-rich plasma or washed platelets. On the other hand, use of both signals is helpful when analysis is being performed on a heterogeneous sample containing all blood cell elements. The forward scatter gate is
[36]
PLATELET ANALYSIS BY FLOW CYTOMETRY
r,-w2001 rn ~E z:)
z J J w
B
gate IO0-
0
425
2
I
s6o
,ooo
560
-
I00o '
FORWARD SCATTER
FIG. 3. Forward-scatter analysis of resting (A) and ADP-stimulated (B) platelets demonstrating platelet aggregates. Platelets in platelet-rich plasma were stimulated by incubation with 5 ~ M ADP for 20 min prior to formaldehyde treatment. Control platelets were not stimulated, The gate shown in (A) includes 80% of the resting platelets that are 2/zm and greater in diameter. As seen in (B), after ADP stimulation and aggregate formation this same gate includes only 55% of the total number of particles analyzed. The aggregated platelets are represented by that portion of curve (B) that extends beyond the gate and up to channel 1000.
selected to include single platelets and exclude aggregates and platelet fragments. The lower channel is determined by displaying the forwardangle scatter histogram generated by 2-/zm beads, thus excluding platelet fragments. The upper channel, chosen from a point on the forward-scatter histogram of fixed, resting platelets is set so that 80% of the analyzed cells will be contained within the gate. The placement of this channel is based on our analysis of forward-angle scatter histograms generated by platelet preparations that intentionally contain platelet aggregates. These samples are produced by adding 5/xM ADP to PRP prior to formaldehyde treatment (Fig. 3). Fluorescence analysis of platelets that fall within the sizing gate is similar to that used for any other FITC-labeled cell. Excitation of the dye is achieved with an argon ion laser emitting a 488-nm laser beam. We have successfully used lasers with power ratings of 100 mW and 5 W. For these lasers, the corresponding power output at 488 nm is 50 mW and 280-300 mW, respectively. The power output of the laser determines, in part, the sensitivity of the system. For most targets, the power of the exciting laser beam will determine the strength of the excitation signal emitted. If the copy number of the antigen of interest is very low its detection may be enhanced by using a more powerful laser. Direct immunofluorescent
426
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
[36]
staining of antigens with FITC-labeled primary antibodies may also be facilitated by higher power lasers. With the platelets diluted to 50,000/zl we adjust the sample flow rate so that the cells are analyzed at a rate of 300 to 500/sec. The fluorescence signal can be subjected to either linear or logarithmic amplification. Linear amplification is best suited for signals that vary over only a small range. Logarithmic amplifiers are better suited for analysis of data that vary over a wide range of fluorescence intensity. Detection of subpopulations, particularly if they are very much brighter or dimmer than the majority of cells, will be aided by logarithmic amplification.I° Data Analysis and System Calibration Because current methods in flow cytometry do not permit direct quantitation of cell surface antigens, results are usually expressed in arbitrary units. Often these units refer to the peak or mean channel number describing the fluorescence histogram of a cell population. Fluorescence density calculations based on individual cell fluorescence intensity (channel number) divided by its corresponding 90 ° scatter channel number may also be utilized. To compare results from one experiment to another and from day to day, it is necessary to have a method for instrument calibration. We have found that commercially available beads are too bright and prefer using glutaraldehyde-fixed chicken erythrocytes. Using the fluorescence photomultiplier gain control we locate the erythrocyte fluorescence histogram so that peak fluorescence is located midway along the fluorescence intensity axis. Similarly, we have also utilized Fluorotrol GF (a research reagent prepared by Ortho Diagnostic Systems, Westwood, MA) for machine calibration. Fluorotrol GF is a mixture of thymocyte nuclei in which approximately 20% of the nuclei are unstained, 40% are stained with 50,000 FITC molecules per nucleus, and 40% are stained with 220,000 FITC molecules per nucleus. Depending on the laser power of a specific instrument, fluorescence analysis of Fluorotrol GF will produce a two- or threepeak histogram (in low-powered systems autofluorescence from the unstained nuclei may not be detected). The fluorescence peaks can be aligned over the same channels each day and thus the machine repeatedly calibrated. Analysis of Platelets without Fixation and in Whole Blood As mentioned previously, the effect of formaldehyde fixation on a specific antigen must be determined experimentally. Saunders et al. reported that the distribution of the Zw a antigen is altered by fixation. ~5Other f5 p. W. G. S a u n d e r s , B. E. Durack, and H. K. Narang, Br. J. Haematol. 62, 631 (1986).
[36]
PLATELET ANALYSIS BY FLOW CYTOMETRY
427
investigators have used flow cytometry techniques to examine intrinsic or adsorbed platelet antigens without prior platelet fixation. NicholsonWeller e t al. have described the presence of decay-accelerating factor, a complement regulatory substance, on the surface of platelets. 9 They utilized washed platelets for their studies and analyzed them shortly after preparation. Dunstan and Simpson have used flow cytometry to analyze platelet content of ABH, Ii, Lewis, P, PL A1, Bak, and HLA class I antigens. 8 In their studies platelets were immunostained after washing and then formaldehyde fixed. Jennings and others reported on the analysis of the platelet glycoprotein IIb-IIIa complex in dilute PRP or whole blood. 3 To keep the platelets from aggregating during antibody staining and analysis they mixed previously anticoagulated PRP or whole blood with an equal volume of a buffer containing prostacyclin (0.154 M NaC1, 0.01 M Tris, 0.0005 M Ca2C1, 50 /~M prostacyclin). The applicability of this method for analysis of GPIb has not been evaluated. Moake et al. reported that prostacyclin can inhibit vWF-dependent platelet agglutination by altering the platelet surface. 16 Although prostacyclin did not block vWF binding to platelets, it is not known whether GPIb immunoreactivity is altered. Other studies have focused on identification of circulating activated platelets using flow cytometry techniques. Specific surface antigens have been identified that predict the activated state. 17.18Similarly, the presence of platelet-derived microparticles has been associated with intravascular platelet activation. 19.20 Acknowledgments B. Adelman is the recipient of NHLBI Clinical Investigator Award HL01053. Additional support was provided by a grant from the Council for Tobacco Research U.S.A., Inc.
t6 j. L. Moake, S. S. Tang, J. D. Olson, J. H. Troll, P. L. Cimo, and P. J. A. Davies, Am. J. Physiol. 241, H54 (1981). 17 C. L. Berman, E. L. Yeo, J. D. Wencel-Drake, B. C. Furie, M. H. Ginsberg, and B. Furie, J. Clin. Invest. 78, 130 (1986). 18 S. J. Shattil, M. Cunningham, and J. A. Hoxie, Blood 70, 307 (1987). 19 C. S. Abrams, N. Ellison, A. Z. Budzynski, and S. J. Shattil, Blood 75, 128 (1990). 2o j. N. George, E. B. Pickett, S. Saucerman, R. P. McEver, T. J. Kunicki, N. Kieffer, and P. J. Newman, J. Clin. Invest. 78, 340 (1986).
PLATELET
RECEPTORS"
ASSAYS AND PURIFICATION
[36]
[36] E v a l u a t i o n o f P l a t e l e t S u r f a c e A n t i g e n s b y F l u o r e s c e n c e Flow Cytometry B y B U R T A D E L M A N , PATRICIA
CARLSON,
a n d ROBERT I. H A N D I N
Introduction Fluorescence flow cytometry has proven to be a useful technique for identifying cell surface-associated antigens. Large numbers of cells can be examined simultaneously and more than one property of each cell analyzed, thereby facilitating the identification of subpopulations. Both intrinsic constitutive membrane proteins and molecules adsorbed or bound to the cell surface can be identified. For example, by using antibodies directed against unique surface markers this method has been used to identify T lymphocyte subtypes within heterogeneous leukocyte preparations. 1 This chapter will focus on methods for the analysis of platelets by fluorescence flow cytometry. As an example, we describe the identification of glycoprotein Ib (GPIb), the platelet receptor for von Willebrand factor (vWF),2 by incubation of platelets with anti-GPIb antibody. Other investigators have used similar methods to study the glycoprotein IIb-IIIa complex 3 and platelet-associated immunoglobulin, 4-6 complement, 7 blood group antigens,8 and decay-accelerating factor. 9 The technique of fluorescence flow cytometry can also be used to study the binding of proteins like vWF, fibrinogen, and fibronectin to the platelet surface. In addition, the method can be used to screen monoclonal and polyclonal antibodies directed against platelet antigens. For a complete discussion of the theory and instrument design underlying the development of fluorescence flow cytometry equipment and
I E. L. Reinherz and S. F. Schlossman, Cell (Cambridge, Mass.) 19, 821 (1980). 2 B. Adelman, A. D. Michelson, R. I. Handin, and K. A. Ault, Blood 66, 423 (1985). 3 L. K. Jennings, R. A. Ashmun, W. C. Wang, and M. E. Dockter, Blood 68, 173 (1986). 4 j. Lazarchick and S. A. Hall, J. Immunol. Methods 87, 257 (1986). 5 j. Lazarchick, P. V. Genco, S. A. Hall, A. D. Ponzio, and N. M. Burdash, Diagn. lmmunol. 2, 238 (1984). 6 C. S. Rosenfeld and D. C. Bodensteiner, Am. J. Clin. Pathol. 85, 207 (1986). 7 V. Martin, K. A. Ault, and R. I. Handin, Blood 54, Suppl. 1, 112 (1979). 8 R. A. Dunstan and M. B. Simpson, Br. J. Haematol. 61, 603 (1985). 9 A. Nicholson-Weller, J. P. March, C. E. Rosen, D. S. Spicer, and K. F. Austen, Blood 65, 1237 (1985).
METHODS IN ENZYMOLOGY, VOL. 215
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
[36]
PLATELET ANALYSIS BY FLOW CYTOMETRY
421
TABLE I PREPARATION OF PLATELETS FOR ANALYSIS BY FLUORESCENCE FLOW CYTOMETRY
1. 2. 3. 4. 5. 6. 7. 8.
Prepare platelet-rich plasma Fix platelets in 2% (v/v) formaldehyde Wash fixed platelets Incubate platelets with primary or control antibody Wash platelets free of excess antibody Incubate platelets with fluorescein-labeled second antibody Wash platelets free of excess antibody Analyze by flow cytometry
m e t h o d s the reader is referred to a review l° and two texts 11'12 on this subject. Preparation of Platelets and Fluorescent Staining We routinely treat platelets with formaldehyde 13 prior to incubation with antibody (see Table I). A major advantage of formaldehyde fixation is inhibition of degrading e n z y m e s present within platelets that might r e m o v e surface antigens. This is a particular problem with G P I b , as it is rapidly released f r o m the platelet surface by a calcium-dependent protease. ~4Fixation also prevents platelet aggregation during washing steps and enhances the stability of the stained cells. We find that formaldehydetreated platelets can be stored at 4 ° for up to 5 days prior to analysis with minimal loss of the fluorescent signal. The effect of fixation on the immunoreactivity, platelet content, and distribution of any antigen studied must be determined b y the investigator. If fixation alters the i m m u n o r e a c tivity of a particular antigen it is possible to treat platelets with formaldehyde after immunostaining. 8 Whole blood is drawn through a 21-gauge butterfly-type needle into a plastic syringe containing 3.8% (w/v) sodium citrate. Nine parts of blood are mixed with one part anticoagulant using a double-syringe technique. First draw 3 ml of blood into a syringe that is then discarded. A second syringe containing sodium citrate is used to draw the blood. The anticoagu10D. R. Parks and L. A. Herzenberg, this series, Vol. 108, p. 197. 11M. A. Van Dilla, P. N. Dean, O. D. Laerum, and M. R. Melamed, "Flow Cytometry: Instrumentation and Data Analysis." Academic Press, London, 1985. 12H. M. Shapiro, "'Practical Flow Cytometry." Alan R. Liss, New York, 1985. 13j. p. Allain, H. A. Cooper, R. H. Wagner, and K. M. Brinkhous, J. Lab. Clin. Med. 85, 318 (1975). 14D. R. Phillips and M. Jakfibovfi, J. Biol. Chem. 253, 3435 (1978).
422
P L A T E L E T RECEPTORS: ASSAYS A N D P U R I F I C A T I O N
[36]
lated blood is mixed and then centrifuged in polypropylene test tubes at 200 g for 10 min at room temperature. If blood is obtained from a normal donor, 15-30 ml will provide an adequate number of platelets for study although studies have been performed on as little as 1 ml of whole blood. The upper two-thirds of the platelet-rich plasma (PRP) is collected and mixed with an equal volume of buffered 2% (v/v) formaldehyde that has been warmed to 37°. Buffered formaldehyde (2% formaldehyde in TBS: 10 mM Tris, 0.01 M NaC1, pH 7.4) is kept refrigerated and made fresh every week from a stock solution of 37% formaldehyde (Fisher Scientific Co., Fair Lawn, N J). After incubation for 30 min at 37° the platelets are centrifuged at 3200 g for 10 min at 4°, resuspended in washing buffer (TBS containing 2 mM EDTA), and washed twice by centrifugation at 3200 g for 10 min at 4°. After washing, the platelets are suspended in TBS without EDTA. If staining and analysis are not done immediately the platelets should be stored at 4° in TBS containing 0.02% sodium azide. Prior to use, previously stored platelets should be centrifuged and resuspended in fresh buffer. Although we have used TBS in this procedure other physiologic buffers should be equally effective.
Immunofluorescent Staining Glycoprotein Ib was detected on formaldehyde-fixed platelets by indirect immunofluorescent staining (Fig. 1). In our studies we have used both monoclonal and polyclonal antibodies directed against the a chain of GPIb. Monoclonal antibodies were produced by immunizing mice with intact platelets or with purified glycocalicin, a proteolytic fragment of the a chain of GPIb (see [25] in this volume). Polyclonal antibodies were produced by immunizing rabbits with glycocalicin. The second agent is always a fluorescein isothiocyanate (FITC)-conjugated, affinity-purified, F(ab')2 fragment of appropriate specificity (Cooper Biomedical, Inc., Malvern, PA). Formaldehyde-fixed platelets in TBS were diluted to a concentration of 50,000 platelets//.d. They were then incubated for 30 min at 4° with primary antibody (antibody may be in purified form or used as ascites or serum). After incubation the platelets were washed three times by centrifugation (3200 g for 15 min at 4 °) and resuspended in TBS at the original volume. The platelets are then incubated with the appropriate FITC-labeled second antibody for 30 min at 4° and then again washed three times as mentioned above. Each antibody should be used at a saturating concentration, which must be determined by trial and error.
[36]
PLATELET ANALYSIS BY FLOW CYTOMETRY
423
,,,°::200]
03
D IO0-
CONTROL ANTIBODY
Z _1 _.1 W (.)
0
5OO 10~30 FLUORESCENCEINTENSITY
FIG. 1. Fluorescence flow cytometry analysis of GPlb on formaldehyde-treated platelets. Platelets were prepared as described in text and stained with 3G6, a monoclonal antibody directed against the a chain of GPIb or with a control, nonspecific antibody. Fluorescence analysis was performed on an Ortho 50H flow cytometer equipped with a 100-mWargon ion laser operating at 488 nm and 50 mW. Fluorescence intensity is displayed on a linear scale in which the scale units are channel numbers. Each curve represents 10,000 platelets. Both the mean and peak fluorescence signal from the specifically stained platelets is greater than that of the nonspecifically stained platelets.
Fluorescence Flow C y t o m e t r y All flow c y t o m e t e r s currently marketed utilize similar technology for sample presentation, illumination, and raw data capture. Differences exist in the number, power, and placement of light sources and in capabilities for data analysis and presentation. The methods described here should be applicable to machines with even the most basic configuration. Some flow cytometers use a mercury arc lamp rather than a laser as the source of illumination. We have not had adequate experience with such machines to c o m m e n t on their use, so that this discussion is based on experience only with c y t o m e t e r s equipped with a laser. Identification of specific cell populations within a sample prior to fluorescence analysis is usually determined by light scatter measurements. Forward-angle light scatter is proportional to overall cell size, while 90 ° scatter is related to internal cell structure. The two scatter signals taken together can be used to distinguish different cell types in mixed samples (such as whole blood), and to identify dead cells in homogeneous cell preparations. Once a cell population or subpopulation is identified by
424
PLATELET RECEPTORS" ASSAYS AND PURIFICATION
2OO"
[36]
gore I
n~ W m
2-~m B E A D S
:D IOO
Z
.-I _.1 W t_)
360
680
FORWARD SCATTER
FIG. 2. Determination of the appropriate sizing gate for analysis of platelets by flow cytometry. This composite picture includes the forward-anglescatter analysis of 2-p.mbeads and formaldehyde-treated resting platelets. The gate is set to include all particles falling within the region indicated. This region will include approximately80% of the resting platelets that are 2/zm and greater in diameter. Each curve represents 10,000platelets and is displayed on a linear scale in which the scatter units are channel numbers.
its light scatter signal (forward scatter alone or forward- and right-angle scatter), a gate can be drawn that will segregate the population of interest and the machine instructed to collect fluorescent signals from all particles whose scatter characteristics fall within the defined gate. Gate boundaries are delimited by user-defined upper and lower channels. Light scatter from nonspherical cells, such as platelets, is affected by the orientation of each target cell flowing past the laser beam. Nonspherical cells tend to orient with their long axes parallel to the direction of flow. E v e n aligned in this manner, platelets will pass through the laser beam in various orientations and produce a range of scatter signals because of their discoid shape. In addition, aggregated platelets will produce varying scatter signals depending on aggregate size and orientation, We use forward light scatter measurements derived from 2-/xm beads (Polysciences, Warrington, PA) and formaldehyde-treated resting platelets to develop a sizing gate prior to analysis of fluorescent labeled platelets (Fig. 2). We have not found that concurrent 90 ° scatter adds significantly to this process when analyzing a homogeneous sample derived from platelet-rich plasma or washed platelets. On the other hand, use of both signals is helpful when analysis is being performed on a heterogeneous sample containing all blood cell elements. The forward scatter gate is
[36]
PLATELET ANALYSIS BY FLOW CYTOMETRY
r,-w2001 rn ~E z:)
z J J w
B
gate IO0-
0
425
2
I
s6o
,ooo
560
-
I00o '
FORWARD SCATTER
FIG. 3. Forward-scatter analysis of resting (A) and ADP-stimulated (B) platelets demonstrating platelet aggregates. Platelets in platelet-rich plasma were stimulated by incubation with 5 ~ M ADP for 20 min prior to formaldehyde treatment. Control platelets were not stimulated, The gate shown in (A) includes 80% of the resting platelets that are 2/zm and greater in diameter. As seen in (B), after ADP stimulation and aggregate formation this same gate includes only 55% of the total number of particles analyzed. The aggregated platelets are represented by that portion of curve (B) that extends beyond the gate and up to channel 1000.
selected to include single platelets and exclude aggregates and platelet fragments. The lower channel is determined by displaying the forwardangle scatter histogram generated by 2-/zm beads, thus excluding platelet fragments. The upper channel, chosen from a point on the forward-scatter histogram of fixed, resting platelets is set so that 80% of the analyzed cells will be contained within the gate. The placement of this channel is based on our analysis of forward-angle scatter histograms generated by platelet preparations that intentionally contain platelet aggregates. These samples are produced by adding 5/xM ADP to PRP prior to formaldehyde treatment (Fig. 3). Fluorescence analysis of platelets that fall within the sizing gate is similar to that used for any other FITC-labeled cell. Excitation of the dye is achieved with an argon ion laser emitting a 488-nm laser beam. We have successfully used lasers with power ratings of 100 mW and 5 W. For these lasers, the corresponding power output at 488 nm is 50 mW and 280-300 mW, respectively. The power output of the laser determines, in part, the sensitivity of the system. For most targets, the power of the exciting laser beam will determine the strength of the excitation signal emitted. If the copy number of the antigen of interest is very low its detection may be enhanced by using a more powerful laser. Direct immunofluorescent
426
PLATELET RECEPTORS: ASSAYS AND PURIFICATION
[36]
staining of antigens with FITC-labeled primary antibodies may also be facilitated by higher power lasers. With the platelets diluted to 50,000/zl we adjust the sample flow rate so that the cells are analyzed at a rate of 300 to 500/sec. The fluorescence signal can be subjected to either linear or logarithmic amplification. Linear amplification is best suited for signals that vary over only a small range. Logarithmic amplifiers are better suited for analysis of data that vary over a wide range of fluorescence intensity. Detection of subpopulations, particularly if they are very much brighter or dimmer than the majority of cells, will be aided by logarithmic amplification.I° Data Analysis and System Calibration Because current methods in flow cytometry do not permit direct quantitation of cell surface antigens, results are usually expressed in arbitrary units. Often these units refer to the peak or mean channel number describing the fluorescence histogram of a cell population. Fluorescence density calculations based on individual cell fluorescence intensity (channel number) divided by its corresponding 90 ° scatter channel number may also be utilized. To compare results from one experiment to another and from day to day, it is necessary to have a method for instrument calibration. We have found that commercially available beads are too bright and prefer using glutaraldehyde-fixed chicken erythrocytes. Using the fluorescence photomultiplier gain control we locate the erythrocyte fluorescence histogram so that peak fluorescence is located midway along the fluorescence intensity axis. Similarly, we have also utilized Fluorotrol GF (a research reagent prepared by Ortho Diagnostic Systems, Westwood, MA) for machine calibration. Fluorotrol GF is a mixture of thymocyte nuclei in which approximately 20% of the nuclei are unstained, 40% are stained with 50,000 FITC molecules per nucleus, and 40% are stained with 220,000 FITC molecules per nucleus. Depending on the laser power of a specific instrument, fluorescence analysis of Fluorotrol GF will produce a two- or threepeak histogram (in low-powered systems autofluorescence from the unstained nuclei may not be detected). The fluorescence peaks can be aligned over the same channels each day and thus the machine repeatedly calibrated. Analysis of Platelets without Fixation and in Whole Blood As mentioned previously, the effect of formaldehyde fixation on a specific antigen must be determined experimentally. Saunders et al. reported that the distribution of the Zw a antigen is altered by fixation. ~5Other f5 p. W. G. S a u n d e r s , B. E. Durack, and H. K. Narang, Br. J. Haematol. 62, 631 (1986).
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PLATELET ANALYSIS BY FLOW CYTOMETRY
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investigators have used flow cytometry techniques to examine intrinsic or adsorbed platelet antigens without prior platelet fixation. NicholsonWeller e t al. have described the presence of decay-accelerating factor, a complement regulatory substance, on the surface of platelets. 9 They utilized washed platelets for their studies and analyzed them shortly after preparation. Dunstan and Simpson have used flow cytometry to analyze platelet content of ABH, Ii, Lewis, P, PL A1, Bak, and HLA class I antigens. 8 In their studies platelets were immunostained after washing and then formaldehyde fixed. Jennings and others reported on the analysis of the platelet glycoprotein IIb-IIIa complex in dilute PRP or whole blood. 3 To keep the platelets from aggregating during antibody staining and analysis they mixed previously anticoagulated PRP or whole blood with an equal volume of a buffer containing prostacyclin (0.154 M NaC1, 0.01 M Tris, 0.0005 M Ca2C1, 50 /~M prostacyclin). The applicability of this method for analysis of GPIb has not been evaluated. Moake et al. reported that prostacyclin can inhibit vWF-dependent platelet agglutination by altering the platelet surface. 16 Although prostacyclin did not block vWF binding to platelets, it is not known whether GPIb immunoreactivity is altered. Other studies have focused on identification of circulating activated platelets using flow cytometry techniques. Specific surface antigens have been identified that predict the activated state. 17.18Similarly, the presence of platelet-derived microparticles has been associated with intravascular platelet activation. 19.20 Acknowledgments B. Adelman is the recipient of NHLBI Clinical Investigator Award HL01053. Additional support was provided by a grant from the Council for Tobacco Research U.S.A., Inc.
t6 j. L. Moake, S. S. Tang, J. D. Olson, J. H. Troll, P. L. Cimo, and P. J. A. Davies, Am. J. Physiol. 241, H54 (1981). 17 C. L. Berman, E. L. Yeo, J. D. Wencel-Drake, B. C. Furie, M. H. Ginsberg, and B. Furie, J. Clin. Invest. 78, 130 (1986). 18 S. J. Shattil, M. Cunningham, and J. A. Hoxie, Blood 70, 307 (1987). 19 C. S. Abrams, N. Ellison, A. Z. Budzynski, and S. J. Shattil, Blood 75, 128 (1990). 2o j. N. George, E. B. Pickett, S. Saucerman, R. P. McEver, T. J. Kunicki, N. Kieffer, and P. J. Newman, J. Clin. Invest. 78, 340 (1986).
