A n Analysis of Platelet Activation and Aggregation Produced by Three Classes of Contrast Media1 Karen L. Hay, MS, MT(ASCPj2 PURPOSE: To evaluate the platelet activation and aggregation pro- Brian 5. Bull, MD2 duced by ionic high-osmolality contrast media (HOCM) and both

ionic and nonionic low-osmolality contrast media (LOCM). MATERIALS AND METHODS: After each agent was mixed with hep-

Index terms: Blood, platelets Con- arinized blood, sequential platelet counts were used to monitor trast media, comparative studies Con- trast media, complications * Contrast me-

platelet aggregation, and flow cytometry was used to monitor both dia, experimental studies aggregation and activation. Aggregation was measured with CD4la-

FITC (specific for glycoprotein 1%-IIIa) and activation was mea- -1995; 6:211-217 sured with CD62-PE (specific for P-selectin). Abbreviations: FITC = fluorescein RESULTS: High concentrations of the nonionic LOCM (>31% by vol- isothiocyanate, HOCM = high-osmolality ume in whole blood) induced more than 90% platelet activation 'Ontrast media, = low-osmolality within 2 minutes of exposure to freshly drawn heparinized whole contrast media

blood. Aggregation followed immediately after activation and was somewhat reversible. High concentrations of the ionic HOCM (>31%) induced prominent activation, although it occurred at a much slower rate and to a lesser degree than with the nonionic media. There was approximately 70% activation after 45 minutes of expo- sure. Ionic HOCM inhibited platelet aggregation, however. The ionic LOCM ioxaglate produced minimal or no platelet aggregation or ac- tivation. CONCLUSION: There is likely to be an agent-related difference in the risk of platelet activation and aggregation in the catheter lumen and in the immediate environment of the catheter tip, where high concentrations of contrast media are known to exist.

R m I o c R A p H I c contrast media have tems (eg, vascular, renal, pulmonary, been in use since sodium-iodide solu- cardiac, cerebral), and most of them tion was first introduced for urogra- have been attributed to the high 0s- phy procedures in 1923 (1). Because molality and ionic nature of the me- of the considerable toxicity of these dia. early agents, alternatives have been In an attempt to reduce these side explored, which has led to the devel- effects, a number of low-osmolality opment of the modern diatrizoates contrast media (LOCM) have re- and iothalamates. These media have cently been developed. There are two

From the Department of Pathology and osmolalities in the vicinity of 1,400- classes of LOCM because two ap- Laboratory Medicine, Linda Univer- 2,000 mosmkg, with three iodine at- proaches were used to reduce the 0s- sity Medical Center, 11234Anderson s t , Oms for each pair of osmotically ac- molality. The first approach involved Lorna Linda, CA 92354. Received April 14, tive particles in solution (2). Al- the replacement of the ionic carboxyl 1994;revisi0nrequested June krevision though considerably less toxic than group and noniodine side chains with received September 19; accepted Septem- ber 27. This study is supported by the original media used, these ionic hydrophilic organic side chains. The Mallinckrodt Medical, s t . Louis, MO. ~ d - high-osmolality contrast media resulting agents, nonionic LOCM, dress reprint requests to B.S.B. (HOCM) are still associated with a are, as the name implies, nonionic

have the exist- variety of adverse reactions that can and have osmolalities in the vicinity ence of a potential conflict of interest. be mild, moderate, or severe. These of 600-700 mosmkg, which is less o SCVIR, 1995 reactions affect different organ sys- than half that of the HOCM (2).

21 1

212 Journal of Vascular and Interventional Radiology

March-April 1995

The other approach involved de- velopment of an ionic dimer by the polymerization of two triiodinated benzoic acid derivatives to form a single anion. The resulting agent, sodium meglumine ioxaglate, has six iodine atoms for every two osmotic particles; thus the osmolality is sub- stantially reduced, and the iodine content is maintained. Although it is still ionic, its osmolality is only 600 mosmlkg, hence the category "ionic LOCM (2).

Although death rates do not seem to differ with use of the LOCM or the HOCM, virtually all investiga- tors have observed fewer severe ad- verse reactions with the LOCM (3). Two aspects of this matter remain unclear. First, it is not known whether the lower frequency of side effects exhibited with LOCM is due to their lowered osmolality or to some other factor. And second, there are reports that nonionic LOCM may be associated with a higher fre- quency of thrombosis than ionic me- dia (either HOCM or LOCM) (4,5).

The effects of these three classes of contrast media on blood clotting have been studied by several inves- tigators (4,6-12). We report their ef- fects on platelet function.

MATERIALS AND METHODS

For the HOCM, we studied dia- trizoate-370 (Renografin-76; Squibb, New Brunswick, NJ, and MD-76; Mallinckrodt, St. Louis, Mo). For the ionic LOCM, we studied ioxaglate- 320 (Hexabrix; Mallinckrodt). Three nonionic LOCM were examined: ioversol-320 (Optiray; Mallinckrodt), iohexol-300 (Omnipaque; Sanofi Winthrop, New York, NY), and iopamidol-300 (Isovue; Squibb).

Blood specimens were collected from healthy adult volunteers who were not known to have ingested as- pirin or other platelet-active medica- tion within the preceding 10 days. Blood samples were drawn with cleared 21-gauge butterfly blood col- lection sets into plastic syringes that were preheparinized with measured

Figure 1. Quad- rant locations of the various platelet populations when studied with flow cytometry with CD4la-FITC (alate- let marker) a i d

CD62-PE CD62-PE (activation (Activation marker). Platelets Marker, had been ~reviouslv selected b;r means of gating on size (for- ward scatter) and GpIIb-IIIa positivity (CD4la-FITC).

Activated Aggregated Activated

Resting I

Platelets I Aggreqated

I I

CD4la-FITC (Platelet Marker)

amounts of pork gut-derived sodium heparin (Lyphomed, Rosemont, Ill, or SoloPak Laboratories, Franklin Park, Ill). The final concentration of heparin was 4 U(USP)/mL. The con- trast agents were evaluated at con- centrations of up to 40% by volume in whole blood. Although these con- centrations might at first seem ex- cessively high, it is standard prac- tice to infuse contrast agents at con- centrations of 100%. In the lumen of the catheter and in the vicinity of the catheter tip, concentrations of 40% or more would be expected to be present routinely. Two methods were used to evaluate the effects of the three classes of contrast media.

Sequential Platelet Counts to Measure Platelet Aggregation Freshly drawn heparinized blood

(2.5 mL) was immediately dispensed into glass tubes that contained 750 pL of the following agents: ioversol (n = 12), ioxaglate (n = 6), or diatri- zoate (MD-76, n = 7; Renografin-76, n = 6). Single tests with iopamidol and iohexol were also performed to determine whether other nonionic agents reacted similarly to ioversol. The concentration of contrast agent in each tube was 23%. Complete blood cell counts were obtained im- mediately and at intervals of 5-10 minutes during a period of 1 or more hours with a Sysmex NE-8000 he-

matology analyzer (Sysmex, Long Grove, Ill). Because this instrument transports, mixes, and aspirates samples automatically, the mixing and handling of tubes was a con- stant. The sequential measurements were monitored for a rapid decrease in platelet count (which would indi- cate aggregation) immediately after the blood and contrast agent were mixed, potentially followed by an in- creased platelet count (which would indicate deaggregation).

Flow Cytometry to Measure Platelet Activation and Aggregation Freshly drawn heparinized blood

(2 mL) was dispensed into polysty- rene tubes that contained premea- sured amounts of the following: ioversol (n = 61, iopamidol (n = 41, iohexol (n = 3), ioxaglate (n = 7), dia- trizoate (MD-76, n = 6; Renografin- 76, n = 5), or 0.9% sodium chloride (n = 3). Final concentrations of con- trast media or sodium chloride were 5%, 17%, 31%, and 40%. Heparin- ized blood with no contrast media added (the "heparin blank") was as- sayed concurrently with each mix- ture of blood and contrast agent. At 30 seconds and at intervals (1, 2,5, 10,15,25,35,45 minutes) during the next 45 minutes, approximately 50-pL aliquots were removed and fixed in 1 mL of 2% paraformalde-

Hay and Bull 213

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0 ! I I I I I I I I I I I 1 I I I

0 30 60 90 120 150

Time post-collection (min)

+loxaglate -a- Diatrizoate +Diatrizoate (Hexa brix) (MD-76) (Renografin-76)

-e- lohexol -x- loversol -c- lopamidol (Omnipaque) (Optiray-320) (lsovue-300)

Figure 2. Effects of concentrations of 23% of various contrast media on the platelet counts of heparinized blood (4 UImL). The mixtures that contained nonionic contrast media showed full platelet aggregation by the time the first platelet count was com- pleted at approximately 7 minutes. Subsequent measurements, which showed in- creasing platelet counts, illustrate the recovery phase as the platelets deaggregate. Dashed lines indicate presumed changes in platelet count between the exposure to contrast agent at time "0" and the time of the initial platelet count.

hyde (Fisher Scientific, Pittsburgh, Pa) in Dulbecco's phosphate-buff- ered saline (PBS) (Gibco Life Tech- nologies, Grand Island, NY). This fixation halted any platelet activa- tion or aggregation a t that point in time and allowed for analysis of the rapidity with which platelets re- sponded to the agent being evalu- ated. After fixation for a t least 1 hour, the samples were stained for same-day flow cytometric analysis by means of a modification of the method described by Ault et a1 (13,14).

Two stains were used. The first, CD4la-fluorescein isothiocyanate (FITC) (Immunotech, Westbrook, Me), reacted with the platelet mem- brane glycoprotein IIb-IIIa (GpIIb- IIIa) and was used as a platelet marker. The use of this platelet-spe-

cific marker and the differences in size allowed us to recognize and analyze platelets even in the pres- ence of leukocytes and erythrocytes. Thus we were able to perform flow cytometry on whole blood samples rather than on lysed preparations. The second stain, CD62-PE (Becton Dickinson, San Jose, Calif), bound specifically to platelet selectin (P- selectin) on the platelet alpha-gran- ule membrane. These sites are not present on the platelet membrane unless the platelet has been acti- vated and fusion of the alpha-gran- ule membrane with the platelet membrane has occurred. Expression of CD62-PE on the exterior of plate- lets was therefore used as a marker of platelet activation. Staining was performed according to the manufac- turer's instructions. Isotype controls

were stained in parallel with the CD62-PE. Specimens were then as- sayed on the FACScan (Becton Dickinson); a log scale was used for all parameters.

Five thousand "events" were col- lected in a gated region defined ac- cording to size (forward scatter) and the presence of GpIIb-IIIa (CD4la- FITC positivity); this method effec- tively eliminated most white and red blood cells as well as most debris from the data acquisition set. Each "event" corresponded to a single cell or cell clump.

These acquired data were subse- quently analyzed with LYSYS I1 (Becton Dickinson) software. After a line was drawn around the desired platelet population to eliminate as many residual white and red blood cells and as much debris as possible, the platelet population was dis- played in scatterplot form with the CD4la-FITC fluorescence on the ab- scissa and the CD62-PE or isotype control fluorescence on the ordinate.

Each dot on the scatterplot repre- sented a single event. Those dots with low CD4la-FITC fluorescence corresponded to platelets with nor- mal GpIIb-IIIa expression. Dots with high CD4la-FITC fluorescence (CD4la-FITC up-regulation) corre- sponded to clumped platelets with increased GpIIb-IIIa expression per clump, secondary to platelet aggre- gation. In a similar manner, in- creased fluorescence of CD62-PE (up-regulation of P-selectin) on the ordinate was an indicator of platelet activation.

For each timed series of flow cytometry measurements, the initial time was considered the baseline for that series. Quadrants were drawn on that baseline scatterplot such that approximately 97.5% of the data points were included within the lower left quadrant (Fig 1). The in- vestigators were blind to subsequent data obtained in each series at the time the quadrants for each set were defined. Quadrant coordinates were left unchanged throughout the re- mainder of that timed series. By definition, the lower left quadrant

214 Journal of Vascular and Interventional Radiology March-April 1995

contained dots that re~resented resting (nonaggregated and nonacti- vated) platelets. As aggregation or activation occurred in subsequent tubes, portions of the platelet popu- lation shifted into other quadrants. With aggregation there was up-regu- lation of the CD4la-FITC marker for GpIIb-IIIa, and a portion of the platelet population shifted toward the right. With activation there was an increase in CD62-PE expression, and the population shifted upward.

RESULTS

Sequential Platelet Counts At concentrations of 23%, the

ionic media, whether HOCM or LOCM, cause negligible platelet ag- gregation. This result was deduced from the fact that stable platelet counts matching control values per- sisted throughout the entire study (Fig 2). The nonionic LOCM, how- ever, all caused marked and imme- diate platelet aggregation. The ag- gregation was so immediate, in fact, that the decrease in platelet count had already occurred by the time the first blood count was completed (typically within 7-10 minutes after collection). Starting a t 20-30 min- utes, however, the platelet counts in these nonionic LOCM mixtures started to increase again, and the levels approached control values by the end of 2 hours. This gradual re- turn of the platelet count to control levels indicated gradual platelet deaggregation.

Flow Cytometry The ability to rapidly fix aliquots

of the blood-contrast agent mixtures for later flow cytometry allowed for more immediate evaluation of the aggregation phenomenon than could be achieved by means of the plate- let-count method. All preparations started a t the 30-second point with identical, tightly circumscribed, rounded platelet populations that were in the lower left quadrant of the scatterplot, a position which in-

MONOMER (Diatrizoate)

2 min

Dl 5 min

2 min 45 min

CD4la-FITC (PLATELET MARKER

Figure 3. Different patterns of platelet activation and aggregation after exposure of heparinized blood to the three classes of contrast agent. Heparin was present at a concentration of 4 UImL. The contrast agents were present a t a concentration of 40% by volume.

dicated they were predominantly resting platelets. As time pro- gressed, however, four different platelet aggregation and activation patterns developed. The first re- sponse pattern was demonstrated by the heparin blank, heparinized blood that contained no other additive. During a period of 45 minutes, the platelets in the heparin blank showed gradual aggregation fol- lowed by minimal activation (Fig 3, row 1). The second pattern was char- acteristic of nonionic media such as ioversol; in this pattern, activation

preceded aggregation (Fig 3, row 2). The platelet population first shifted from the lower left quadrant to the upper left quadrant, which indicated activation; it only then began to move to the right, which indicated that the activated platelets were un- dergoing aggregation. All nonionic monomers studied followed this pat- tern, and all proved to be potent in- ducers of both aggregation and acti- vation. The third pattern, that of significant activation accompanied by complete absence of aggregation, was demonstrated by both ionic

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0.5 10 20 30 40 50

Time Post-Collection (min)

-+ lopamidol -m-lohexol -&- loversol +Diatrizoate (Isovue) (Omnipaque) (Optiray) (MD-76)

-x- Diatrizoate -o- Heparin + NaCI, 0.9% -A- loxaglate (Renografin-76) Blank (Hexabrix)

Time post-collection (min)

I -40% loversol +31X loversol * 17% loversol I -5% loversol -x-Heparin Blank I

5. Figures 4,5. Time courses of platelet activation (as measured with flow cytom- etry) in the presence of (4) 40% concentration of several contrast media in heparin- ized blood and (5) 0 4 0 % concentration of the nonionic monomer ioversol (Optiray- 320). Results are typical of the other nonionic monomers studied.

monomers (Fig 3, row 3). The fourth pattern was demonstrated by the ionic dimer ioxaglate (Fig 3, row 4). This agent was by far the most in- ert; it produced essentially no acti- vation or aggregation of platelets be- yond that which occurs in otherwise untreated heparinized blood.

A plot demonstrating the time course of platelet activation in typi- cal samples (Fig 4) shows that at the 40% level, all three nonionic con- trast media studied caused almost total activation within 1-2 minutes of exDosure. The diatrizoates Dro- duced more gradual, intermediate levels of activation. The saline con- trol and the ioxaglate mixtures were largely inactive in regard to platelet activation and aggregation.

When several different concentra- tions of the three nonionic LOCM were evaluated, a dose-dependent activation of platelets was found. Figure 5 demonstrates data ob- tained from a characteristic sample treated with ioversol; this pattern is also typical of the effect produced by iopamidol and iohexol.

When isotonic saline and the various contrast agents were evalu- ated a t 5% concentration in whole blood, there was essentially no acti- vation at either 2 or 45 minutes (Table). However, somewhat unex- pectedly, these low concentrations of ioxaglate inhibited activation a t 45 minutes to below that of the heparin blank.

When the same agents were evaluated a t 40% concentration, the results were very different (Table). Although both the nonionic LOCM and the ionic HOCM induced activa- tion as early as 2 minutes after ex- posure, the degree of activation was very different between the two classes. The nonionic agents induced more than 90% activation within 2 minutes of exposure and more than 98% activation at 45 minutes. The ionic HOCM induced approximately 6% activation a t 2 minutes and 70% activation a t 45 minutes. Only sa- line and ioxaglate failed to induce platelet activation. At the 40% con- centration, ioxaglate no longer pro-

216 Journal of Vascular and Interventional Radiology March-April 1995

Percentages of Activated Platelets at 2 and 45 Minutes after Exposure of Blood to Contrast Agent or Saline

Activated Platelets at 2 Minutes Activated Platelets at 45 Minutes Sample

Additive Size Mean SD P* Mean SD P*

None 32 1.42 0.97 . . . 13.03 4.82 . . . 5% Isotonic saline 3 1.26 0.09 >.05 13.18 4.25 >.05 5% Ioversol (Optiray) 6 1.49 0.62 >.05 15.59 9.12 >.05 5% Iopamidol (Isovue) 4 1.24 0.32 >.05 13.31 6.43 >.05 5% Iohexol (Omnipaque) 3 2.66 1.60 >.05 14.72 4.24 >.05 5% Diatrizoate (MD-76) 6 1.34 0.99 >.05 19.20 9.44 >.05 5% Diatrizoate (Renografin) 5 1.42 0.26 >.05 14.37 3.35 >.05 5% Ioxaglate (Hexabrix) 7 1.23 0.61 >.05 8.08 2.46 .014 40% Isotonic saline 3 1.52 0.53 >.05 9.58 3.39 >.05 40% Ioversol (Optiray) 6 93.98 5.03 <.001 98.02 1.25 <.001 40% Iopamidol (Isovue) 4 91.72 7.49 <.001 98.46 0.94 <.001 40% Iohexol (Omnipaque) 3 97.44 1.95 <.001 99.02 0.98 <.001 40% Diatrizoate (MD-76) 6 6.74 3.06 .006 69.81 12.55 <.001 40% Diatrizoate (Renografin) 5 5.57 1.95 .005 70.82 15.93 <.001 40% Ioxaglate (Hexabrix) 7 1.24 0.38 >.05 14.35 7.39 >.05

Note.-Mean and standard deviation (SD) are given as percentages. *P values were determined by means of a comparison with results from samples in which no additives were used.

vided the protective effect demon- strated a t the 5% concentration.

DISCUSSION

Prior work has demonstrated that ionic contrast media function as po- tent anticoagulants that provide protection against thrombosis dur- ing angiographic procedures (5,151. Nonionic media, on the other hand, function as only mediocre anticoagu- lants that allow for the generation of substantial quantities of thrombin in blood-contrast agent mixtures (4). I t is clear from our present work that the three classes of contrast media also differ markedly in their platelet activation and aggregation effects. Somewhat surprisingly, the nonionic agents, which are believed to be the most bland in regard to their biologic effects, are the most powerful of the three in terms of both platelet activation and platelet aggregation. These findings confirm those in the recently published work of Chronos et a1 (16). Using flow cytometry, they demonstrated the profound platelet degranulation brought about by the nonionic agents. To their findings we now add

evidence which indicates that the nonionic agents are capable of caus- ing marked platelet aggregation in addition to activation.

When platelet counts were moni- tored in an evaluation of the aggregational potency of the con- trast media, the three types of non- ionic LOCM produced almost imme- diate platelet aggregation in hep- arinized blood. Ionic media, whether LOCM or HOCM, did not induce platelet aggregation a t any of the concentrations tested.

When evaluated with flow cvto- metric techniques, all three types of nonionic LOCM (at 40% concentra- tion in blood) were capable of caus- ing virtually immediate platelet ac- tivation and aggregation, in that or- der. Whereas more than 90% of the platelets were activated within 2 minutes of exposure to nonionic agents, peak aggregation did not generally occur until approximately 10 minutes after exposure.

Ionic media fell into two classes. The ionic monomers (diatrizoates such as MD-76 and Renografin-76) inhibited aggregation almost com- pletely yet caused substantial acti- vation. The ionic dimer ioxaglate, however, appeared to be almost to-

tally nonstimulatory with regard to both platelet aggregation and activa- tion during the entire 45-minute pe- riod of the study. At low concentra- tions, ioxaglate actually provided a protective effect; i t decreased the baseline activation seen in other- wise untreated heparinized blood.

These findings add to the concern that nonionic LOCM may expose pa- tients undergoing angiography to an increased risk of thrombosis. I t is well known that platelets produce factors that enhance the formation of intrinsic prothrombinase. I t seems possible that a reasonable re- construction of events would show nonionic agents to cause platelet ac- tivation with subsequent adhesion to the wall of the catheter, an event which would result in an environ- ment conducive to clot formation. Al- though only small amounts of blood within the catheter itself or in the immediate vicinity of the catheter tip are likely to be exposed to con- trast agent a t concentrations of 20%-40%, activated platelets on the wall of a catheter are in a position to cause substantial damage; they may initiate clot formation and cause subsequent embolization. Indeed, i t is possible that this is the patho-

Hay and Bull 217

Volume 6 Number 2

physiologic process that underlies the observations of Casalini (17), who used electron microscopy to ex- amine the inner walls of angio- graphic catheters in a human study. He demonstrated clot formation in 60% of patients who were given io- pamidol and in none of the patients given ioxaglate. When clots were ob- served, there was evidence that they were associated with activated platelets. Similar observations were made by Kurisu and Tada (151, who measured the mean weight of clots deposited on guide wires placed in the femoral vessels of dogs. They poted that when ioxaglate was used, clot weight was less than half that present when iopamidol was used.

Although the addition of heparin to nonionic agents may indeed in- crease their anticoagulant potency, our study shows that heparin will in no way prevent or decrease platelet aggregation and activation. Further investigation into the mechanisms involved in these marked differences between types of contrast media is therefore warranted. Any success in attempts to explain why nonionic LOCM promote platelet activation so markedly, even in the presence of heparin, may well lead to the formu- lation of media that are more ideal in this regard.

Some of the available media ex- hibit substantial and probably desir- able anticoagulant properties. Some exhibit essentially no platelet acti- vation o r aggregation. Some media cause minimal side effects. All avail- able media appear to produce satis- factory opacification of blood vessels, and this is the primary reason why they are synthesized. Given these

facts, i t is not unreasonable to hope that it may prove possible to synthe- size different media or to prepare different formulations of older agents that will maximize all of these desirable attributes.

Acknowledgments: We gratefully ac- knowledge Lauralynn Lebeck, PhD, for he? time and assistance in setting up the flow cytometry methodology.

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