Function of human platelets during extracorporeal circulation VINCENT L. HENNESSY, JR,, RICHARD E. HICKS, STEFAN NIEWIAROWSKI, L. HENRY EDMUNDS, JR., AND ROBERT W. COLMAN Departments of Surgery and Medicine, University of Pennsylvania, Philadelphia the Specialized Center for Thrombosis Research, Department of Medicine, Temple 19140 Health Science Center, Philadelphia, Pennsylvania
HENNESSY, VINCENT L., JR., RICHARD E. HICKS, STEFAN NIEWIAROWSKI, L. HENRY EDMUNDS, JR., AND ROBERT W. COLMAN. Function of human platelets during extracorporeal
circuZation. Am. J. Physiol. 232(6): H622-H628, 1977 or Am. J. Physiol.: Heart Circ. Physiol. l(6): H622-H628, 1977. -The interaction between human platelets and nonbiologic surfaces was studied during in vitro recirculation of 500 ml of fresh, heparinized human blood in four different perfusion circuits. Circuits differed in surface area (0.1 m* or 0.9 m*> and in surface composition. No important differences were observed between standard silicone-rubber and filler-free, silicone-rubber surfaces. Platelet counts decreased to 85% of control in 0. lm* circuits, but retained normal sensitivity to aggregating agents and released only small amounts of platelet factor 4 (PF,). In cant rast, platelet counts in 0.9-m* circuits decreased to 20% of control within 2 min and platelet sensitivity was depressed out of proportion to the fall in platelet count. Plasma PF, progressively increased and platelet PF, content progressively decreased during 6 h of recirculation. The results indicate that human platelets may exist in three conditions during extracorporeal circulation. Some platelets are unaltered, some are less sensitive to aggregating agents, and others have undergone extensive release. The ratio of blood volume to surface area appears to be an important determinant of platelet-surface interaction.
19104, and University
study the effects of both surface composition (SSR and FFSR) and surface area on human platelets, and to distinguish reversible and irreversible changes in platelets during extracorporeal perfusion. METHODS
Perfusion circuits. Perfusion circuits with surface areas of 0.1 m2 or 0.95 m2 were assembled from siliconerubber tubing (3/8 in ID, 42 in long, Dow Corning Corp., Midland, Mich. >, silicone-rubber venous reservoirs and spiral-coil oxygenators (Sci-Med Life Systems, Inc., Minneapolis) (Fig. 1). Surface areas were calculated from measured dimensions of the reservoir, tubing, and oxygenator blood contact surfaces. Half of the SSR components were coated and cross-linked with FFSR, Circuits differed by the presence or absence of the spiralcoil-membrane oxygenator (surface areas: membrane, 0.8 m2; cylinder-blood contact surface, 0.05 m2) and by the presence or absence of the FFSR coating. Except for polycarbonate connectors, which represented 3.8 and 0.40% of the surface areas of the O.l-m2 and 0.9-m2 circuits, respectively, all blood contact surfaces were either SSR or FFSR. Smaller circuits of SSR and FFSR filler-free silicone rubber; platelet-surface interaction; platelet were designated 1s and IF, respectively; larger circuits factor 4; platelet aggregation of SSR and FFSR were designated 9s and 9F, respectively. Sci-Med Life Systems, Inc. provided sterile standard WHEN BLOOD CONTACTS a nonbiologic surface, plasma silicone-rubber oxygenators and oxygenators coated and cross-linked with FFSR. Other silicone-rubber compoproteins are adsorbed and platelets adhere (1, 14, 18). During extracorporeal perfusion in the presence of hep- nents for FFSR circuits were filled with a 7% dispersion arin the number of circulating platelets decreases 40- of FFSR in toluene (Sci-Med Life Systems, Inc.). After 60% and the function of remaining platelets is depressed drainage and evaporation of toluene at 23”C, tubing and reservoirs were filled with 100% nitrogen and were irra(19). Deficiency in the number and function of platelets has been suggested as an important cause of bleeding diated at 23°C with 5-10 megarads from a cobalt-60 source (U.S. Army Natick Laboratories, Natick, Mass.). following open-heart operations (16). Efforts to produce a totally nonthrombogenic surface All components were sterilized in ethylene oxide and have not been successful. Recently, Kolobow et al. (13) degassed for at least 72 h before the circuits were assemand Zapol et al. (25) observed that silica particles used bled. Circuit priming. Blood, 500 ml, was drawn from fastas filler to increase the tensile strength of commercial ing donors through siliconized needles and polyvinyl silicone rubber contributes to the loss of platelets during partial cardiopulmonary bypass in lambs. By coating tubing directly into the venous reservoir, which constandard silicone rubber (SSR) of extracorporeal perfu- tained 2,500 U of beef-lung heparin (The Upjohn Comsion systems with filler-free silicone rubber (FFSR) pany, Kalamazoo, Mich.) and 1.650 mg glucose. All which lacks silica particles, they reduced the loss of donors had abstained from all medication for at least 2 platelets. The present experiments were designed to wk prior to their giving blood. H 622
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PLATELET
FUNCTION
DURING
EXTRACORPOREAL
0.1 M2 FIG.
1. Diagrams
0.9 of 0. l-m2
and 0.9-m’
recirculation
CIRCULATION
M2 circuits.
Blood and gas compartments of the 0.9-m2 circuits were flushed with filtered ‘100% carbon dioxide for at least 30 min before priming. Oxygenators were primed by applying a vacuum to the lower gas port and permitting blood to enter the device by gravity. Tubing was filled by gravity. After priming, no gas bubbles were visible in any circuits when recirculation was started. Recirculation system. Blood was recirculated by a precisely shimmed, barely occlusive, calibrated doubleroller pump (Cardiovascular Instrument Corp., Wakefield, Mass.) at a rate equal to twice the remaining circuit blood volume per minute. An adjustable screw clamp maintained circuit back-pressure at 300 mmHg. Blood temperature was maintained at 37°C by immersion of the venous reservoir in a water bath. A mixture of 95% oxygen and 5% carbon dioxide was passed through the gas compartment of the oxygenator at 1 literlmin, countercurrent to the direction of blood flow. Blood was recirculated for 6 h in each circuit on three occasions, for a total of 12 trials. Control samples for platelet studies were obtained directly from the donor (45 ml of blood in 250 U heparin and 165 mg glucose), and were studied immediately and after 1, 3, and 6 h of incubation at 37°C. Samples were taken from the circuit after 2 min of recirculation and after 1, 3, and 6 h of recirculation. Additional samples obtained after 2 min of recirculation were studied after 1, 3, and 6 h of incubation at 37°C. Further samples for platelet counts only were obtained after 15 and 30 min and at 2,4, and 5 h of recirculation. Plasma hemoglobin was measured after 6 h of recirculation in each circuit (2). PlateLet studies. Platelet-rich plasma (PRP) was prepared in plastic ware at 23°C by adding 1 vol of 3.8% sodium citrate to 9 vol of heparinized blood, centrifuging the mixture for 10 min at 170 xg, and gently aspirating the PRP. The platelet count was 300-400,000, unless otherwise specified. The remaining blood was centrifuged for 4 min at 12,000 xg in an Eppendorfcentrifuge
H623 (Brinkmann Instruments, Inc., Westbury, N.Y.) to obtain platelet-poor plasma (PPP), which contained fewer than 1,000 platelets per microliter. Platelets were counted by phase microscopy (4). Platelet aggregation was studied in an aggregometer attached to a strip-chart recorder (Chrono-Log Corp., Havertown, Pa.) with use of a previously described modification (5) of the method of Born (3). PRP was adjusted to 10% light transmission and PPP to 90% light transmission. The extent of aggregation was determined by the maximum percent difference between PRP and PPP achieved in 10 min. As determined previously in 15 normal individuals, complete second-wave aggregation can be assumed (at a 95% confidence level) when PRP light transmittance is greater than 61% for ADP, greater than 57% for epinephrine (unpublished data), and greater than 70% for collagen (5). Because of the apparent log-normal distribution of platelet sensitivities in control subjects, sensitivities were recorded as the log of the concentration of dose of the aggregating agent (5). A second criterion of the second wave of aggregation is the amount of serotonin released from PRP as measured by the method of Jerushalmy and Zucker (11). Normal PRP was incubated with 0.01 vol 14C-labeled serotonin ( [14C]5-hydroxytryptamine, 30 &i/ml; New England Nuclear, Boston). Release was produced by adding respective aggregating agents to labeled PRP stirred at 1,200 rpm at 37°C for 15 min. After centrifugation at 12,000 xg for 4 min, the percentage of 14C-labeled serotonin in the supernatant was expressed as a percentage of labeled serotonin originally taken up. Greater than 20% for ADP, greater than 25% for epinephrine (unpublished data), and greater than 40% for collagen (20) indicated complete second-wave aggregation at a confidence level of 95%. The sensitivity of threshold of platelets to aggregation was defined as the lowest concentration of aggregating agents that produced a complete second wave of aggregation, as judged by aggregometry and 14C-labeled serotonin release. A correlation coefficient of 0.95 between serotonin release and full aggregation was previously demonstrated (20) and was observed in these experiments. Predicted platelet sensitivity curves were constructed for each aggregating agent at low platelet counts by determining platelet sensitivities of PRP from 14 donors which was diluted with the donors’ own respective PPP to platelet counts of 100,000/mm3 and less. In 0.9-m2 circuits, in which immediate thrombocytopenia occurred, a sample of unexposed donor blood was diluted with its own PPP to an equally low platelet count for control platelet function studies. The maximum aggregating doses, even if aggregation failed to occur, were considered to be 50 PM of ADP, 50 ,ug/ml of acid-soluble collagen (5.5 mg/ml; Worthington Biochemical Corp.), and 250 PM epinephrine. Platelet factor 4 (PF,) was measured by the modified radial immunodiffusion method of Mancini et al. (15) with a monospecific antibody against purified PF4, as described by Niewiarowski et al. (17). PF, in plasma was measured after release and centrifugation, as de-
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H624
HENNESSY
scribed for 14C-labeled serotonin. PF, in platelets was measured by determining the total concentration in frozen PRP after exposure to 0.8% Triton X-100 and subtraction of the PF, in PPP. Platelet PF, was expressed in micrograms per log platelets. Scanning electron microscopy. Scanning electron microscopy was used to study differences between SSR and FFSR surfaces before blood contact and after 6 h of recirculation. The perfusion circuit was rinsed with 1 liter Ringer-lactate which contained 1,000 U of beeflung heparin (Upjohn). Random samples of reservoir, tubing, and oxygenator membrane were excised and placed in buffered 3% glutaraldehyde. After fixation, samples were rinsed in distilled water and dehydrated by immersion in increasing concentrations of acetone. Specimens were transferred into liquid carbon dioxide from absolute acetone to which 1 ml of amyl acetate had been added to identify residual organic solvent. Criticalpoint drying was done using liquid CO, in a Denton critical-point dryer (Denton Vacuum Inc., Cherry Hill, N. J. ). Scanning electron microscopy was performed with use of a model JSM 50-A scanning electron microscope from Electron Optic Laboratories Ltd., Tokyo, to obtain qualitative information on adherent platelets. StatisticaL analysis. Observations at each sampling time were considered to be independent of observations at other sampling times to simplify statistical analysis. At each sampling time, mean platelet counts, mean platelet sensitivities, platelet PF, concentrations, plasma PF, concentrations, and plasma hemoglobin concentrations were compared between the four circuits by two-way analysis of variance (22). Since, with one exception, no differences between FFSR and SSR circuits were found, data from FFSR and SSR circuits were combined to compare data from 0. l-m2 and 0.9-m* circuits. The Student paired-t test was used to compare observations within circuits at different sampling times (22) . RESULTS
PZasma hemoglobin. Surface composition did not alter plasma hemoglobin significantly (P > 0.05) after 6 h of recirculation in the 0. l-m2 circuits (Fig. 2). However,
ET AL.
plasma hemoglobin increased significantly when surface area was increased from 0.1 m2 to 0.9 m2 (P < 0.01). PLatelet number. Mean changes in platelet count, expressed as a percentage of the platelet count of blood drawn directly from the donor, are plotted in Fig. 3. In both O.l-m2 circuits, a 10% decrease in platelet count, which was significant (P < 0.05), occurred after 2 min of recirculation. Subsequent platelet counts within circuits remained significantly decreased (P < 0.05) from control counts, but did not differ significantly (P > 0.05) from each other. Platelet counts from 1s circuits did not differ significantly (P > 0.05) from counts from 1F circuits at any sampling time. Platelet counts in both 0.9-m2 circuits fell within 2 min of recirculation to 20% of control (P < 0.01). Over the next 6 h, platelet numbers rebounded to 55% and 41% of control levels in 9s and 9F circuits, respectively. Although the platelet counts in 9s and 9F circuits were not significantly different from each other at any sampling time (P > 0.05), counts at 6 h represented a significant increase from counts after 2 min of recirculation (P < 0.01). Platelet counts from O.l-m2 circuits differed significantly (P < 0.01) from those of 0.9-m2 circuits at all sampling times except control. PLateLet sensitivity. The response of platelets to ADP and epinephrine for each circuit is presented in Fig. 4. In general, data from collagen studies paralleled those from ADP and epinephrine but had wider ranges. Platelet sensitivity to the three aggregating agents did not differ significantly between 1s and 1F circuits at any sampling time. A significant decrease in platelet sensitivity to ADP and collagen occurred after 2 min of recirculation in 0. l-m2 circuits (P < 0.02); however, the initial decrease in platelet sensitivity to epinephrine was not significant (P > 0.05). A more gradual, but significant (P < 0.05), decrease in platelet sensitivity to all three aggregating agents occurred over the next 6 h in recirculated platelets in both 1s and 1F circuits and in platelets of incubated donor blood. However, the changes in platelet sensitivity during recirculation did not differ significantly (P > 0.05) from changes in sensi% of Control Platelet Count
Hb mg/lOOml 250200
-
l50I
1
1
I
i
I
2
3 (hours)
lime
1
4
1
5
J
6
3. Mean changes in platelet count during recirculation in each circuit. Platelet counts are plotted as a percentage of the platelet count of blood drawn directly from donors. o-o, O.l-m2 SSR circuit; o----- o, O.l-m2 FFSR circuit; o-o, 0.9-m2 circuit; l -----0, 0.9-m2 FFSR circuit. Each point is mean of 3 platelet counts. Point i indicates 2 min of recirculation in this and subsequent figures. FIG.
IS FIG.
deviations
2.
Mean after
plasma hemoglobin 6 h of recirculation
IF
9s
9F
concentrations i n each perfusion
with standard circuit.
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PLATELET ADP llM
t
FUNCTION
DURING
EXTRACORPOREAL
FFSR
SSR Cwcuits
1
L
I
I
1
I
L
1
I
3 4 (hours)
5
6
CIRCULATION
Orcults
Epmephrlne 9M
2 Time
I
I
i
I
I
I
I
I
I
23456 Time (hours)
FIG. 4. Lowest mean concentration of ADP and epinephrine required to produce a complete 2nd wave of aggregation as determined by light transmission or serotonin release is presented for blood recirculated in each circuit and for simultaneous control studies. Data from standard silicone-rubber (SSR) circuits appear in left column of graphs, and data from filler-free, silicone-rubber (FFSR) circuits are in right column. oo indicates blood recirculated in 0. l-m2 circuits. l l indicates blood recirculated in 0.9-m2 circuits. clq indicates donor blood incubated at 37°C for 6 h. X-X indicates expected platelet sensitivities for thrombocytopenic samples ( 0.05). Although platelet numbers increased after 6 h of recirculation in 0.9-m* circuits, platelet sensitivity did not improve.
Surface area as opposed to surface composition appeared to be the critical factor in the significant decrease (P < 0.05) in platelet sensitivity to ADP and epinephrine at all sampling times when 0.1-m* and 0.9m2 circuits were compared. Platelet sensitivity to collagen was significantly depressed (P < 0.02) on initial exposure to both 0.1-m* and 0.9-m* circuits; but thereafter, no significant differences (P > 0.05) in platelet sensitivity to collagen occurred as a function of surface area, surface composition, or duration of recirculation. The possibility that observed changes in platelet sensitivity between 0. l-m2 and 0.9-m* circuits may have been due to greater concentrations of ADP or other substances released from hemolyzed red cells was studied. Plasma hemoglobin concentrations between 50 and 300 mg/lOO ml were produced by forcing blood through a no. 25 needle. The blood was then centrifuged at 12,000 x g for 4 min and the hemolyzed plasma obtained was added to PRP prepared from freshly donated blood. Platelet sensitivities to all three aggregating agents were not altered by the addition of this hemolyzed plasma with hemoglobin concentrations up to 300 mg/ 100 ml. The pH in 1s and 1F circuits ranged from 7.36 to 7.41, while that in 9s and 9F circuits ranged from 7.23 to 7.33. To rule out the possibility that changes in pH might affect platelet sensitivity to aggregating agents, an additional control experiment was carried out. The pH of platelet-rich plasma prepared from each of two donors was adjusted to values between 7.12 and 7.37 by bubbled 100% carbon dioxide; the platelet sensitivities to ADP, epinephrine, and collagen were then measured and compared to sensitivities of control aliquots of plateletrich plasma from the same donors. Changes in pH between 7.12 and 7.37 did not affect platelet sensitivities to ADP, epinephrine, and collagen. Similarly, the possibility that increased blood oxygen tension might affect platelet sensitivities possibly by producing reactive intermediates was tested by recirculating fresh donor blood in a 0.9-m* circuit in which the oxygenator was ventilated with 90% nitrous oxide and 10% oxygen. Complete second-wave aggregation as measured by 14C-labeled serotonin release occurred at 47.4 PM t 13.9 for ADP and 54.8 PM t 14.6 for epinephrine (from 2 min to 6 h), and the plotted curves were similar to those for 0.9-m* circuits in Fig. 4. Platelet factor 4 in platelets and plasma. Plasma PF, antigen was less than 0.5 pg/ml plasma in control samples, which is the lower limit of the sensitivity of the method (17). PF, antigen was detected in all circuits after 2 min of recirculation (P < 0.01) (Fig. 5). The increase in both FFSR circuits at 2 min over control samples of unrecirculated blood was less than the increase in SSR circuits, and these differences were significant (P < 0.05). However, after 2 min surface composition did not significantly alter plasma PF,. Plasma PF, increased more rapidly in 0.9-m* circuits and was significantly (P < 0.01) greater than in 0.1-m* circuits at each sampling time after 2 min. Platelet PF, did not change significantly (P > 0.1) in any circuit during the first 3 h of recirculation (Fig. 6). The apparent increase at 2 min in 9F circuits is unex-
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H626
HENNESSY
plained, but is not significant. After 6 h of recirculation, platelets in 0.9-m2 circuits lost most of their PF, content (P < 0.02) and differed significantly (P < 0.01) from PF, contents of platelets recirculated in 0. l-m2 circuits. Surface composition did not significantly (I’ > 0.05) alter PF, content of platelets at any sampling time. Platelet factor 4 values were similar in the 0.9-m2 circuit in which the oxygenator was ventilated with 90% nitrous oxide and 10% oxygen. Under these conditions mean platelet PF, content decreased progressively from 76.5 pg/lOs platelets at 2 min to 30.9 pg/lOs platelets at 6 h. Plasma PF, rose from 4.6 pg/ml at 2 min to 11.7 pug/ ml at 6 h. Scanning electron microscopy. Striking differences in SSR and FFSR surfaces that were not exposed to blood
ET AL.
were not observed, although several photographs suggested that FFSR surfaces were smoother and did not contain protruding silica particles. Qualitative differences in the adhesion of blood elements to SSR and FFSR surfaces were not observed in the random samples of circuit components studied after 6 h of recirculation (Fig. 7). Both single and clumps of platelets and swollen, deformed red cells adhered to both surfaces. Only a few fibrin strands were observed. DISCUSSION
The in vitro recirculation model controls many variables which may affect platelet function during extracorporeal circulation. Fresh heparinized human blood obPF4 J.lg/PltxlO* 200 r 160 I t I ‘1
PF4
ug/ml 24i-
2 3 4 Time (hours)
5
6
Time
(hours)
FIG. 5. Mean changes in plasma platelet factor 4 content during recirculation in each circuit. Symbols are the same as in Fig. 3. Initial values from blood drawn directly from donor were less than 0.5 @g/ml.
FIG. 6. Mean changes in platelet factor 4 content during recirculation in each circuit. Symbols are the same as in Fig. 3. Initial values indicate platelet factor 4 content in blood drawn directly from donors.
FIG. 7. Representative scanning electron micrographs of an SSR oxygenator membrane (A) and an FFSR oxygenator membrane (B) after 6 h of recirculation (x300). P indicates a single platelet; PP indicates a group of platelets. RBC indicates adherent red cells. F
indicates fibrin strands. At high magnification, oxygenator membranes are irregular. No morphologic differences in number and shape of platelets between SSR and FFSR surfaces were seen at magnifications between 100 and 10,000.
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PLATELET
FUNCTION
DURING
EXTRACORPOREAL
H627
CIRCULATION
viates potential extrapolative problems involved in the use of animal platelets. Since animal and human platelets differ in response to aggregating agents (9,21), they may differ in response to foreign surfaces. Absence of an animal in the circuit prevents catabolism of released substances, clearance or sequestration of altered platelets (6), and addition of newly produced platelets to the system. Changes in number and function of platelets and platelet composition can be quantitated. Finally, because platelets are minimally altered in 0.1.m2 circuits, new surface materials can be easily studied by inseries addition to this basic test circuit. The data clearly show that filler-free silicone rubber offers no advantage over standard silicone rubber in the preservation of human platelets during extracorporeal circulation. With the exception of slight differences in plasma PF, after 2 min of recirculation, no differences in platelet numbers, function, or release reaction were observed between the two types of surfaces studied. We conclude that the observed differences noted during partial cardiopulmonary bypass in lambs by Kolobow et al. (13) and Zapol et al. (25) may be related to species differences in platelets or inclusion of animals in the circuit, rather than to surface composition of the bypass circuit. Large numbers of plateletsadhere to the 0.9-m2 surface with first contact. The decrease in platelet count is much greater than that observed in 0.1.m2 circuits. Procedures used in priming oxygenators reduce the likelihood that decreased platelet counts in 0.9-m2 circuits are due to microbubbles (24). Under the same conditions of blood volume and flow rate, the decrease in platelet count appears directly proportional to surface area. Although many potential factors may govern platelet-surface interaction, only flow rate, duration of exposure, and the composition of adsorbed proteins are known to influence platelet adhesion to nonbiologic surfaces (1, 8, 12, 14, 18). In the 0. l-m2 circuits, the ratio of blood to surface area is 1 ml/2 cm2. In 0.95m2 circuits, the ratio is 1 ml/19 cm2. The ratio of an individual’s blood volume (excluding priming fluids) to the surface area of most membrane oxygenator systems is approximately 1 ml/lo-12 cm2. This ratio is consistent with observed changes in platelet number and function during short-term (l-2 h) total cardiopulmonary bypass in humans (7, 16). Consequently, more efficient oxygenators with a reduced surface area may mitigate changes in platelets caused by extracorporeal circulation. Platelet count rose progressively after 1 h of recirculation in 0.9-m2 circuits. This observation clearly indicates that some initially adherent platelets become detached. The progressive rise in platelets correlates with a progressive rise in plasma PF, content and a decrease in mean platelet PF, content to less than 40 pg/lOg platelets. These data indicate that detached platelets have undergone extensive release and are incapable of secondary aggregation and release. Most changes in platelet number and function occur
within 2 min of recirculation. In 0.1.m2 circuits, a 15% decrease in platelet count and a modest increase in plasma PF, occur. Further changes in platelet count, platelet function, and PF, content and release do not occur during the subsequent 6 h. The data do not exclude the possibility that adherent platelets may become detached and may not function, since the experimental error of platelet counts and sensitivity of aggregometry cannot detect the admixture of up to 7% of detached nonfunctioning platelets. The important observation is that the remaining nonadherent platelets function normally and do not react with extracorporeal surfaces. Blood contact surfaces of 0.1-m’ circuits become compatible with platelets and are “inactivated” by contact with 500 ml of fresh, heparinized blood. This observation suggests that platelet-compatible nonbiologic surfaces can be developed. The immediate change in function of nonadherent platelets in 0.9-m2 circuits contrasts with the apparently normal function of the majority of circulating platelets in 0.1.m2 circuits. In 0.9-m2 circuits loss of sensitivity of nonadherent platelets within 2 min is unexplained, but at least two mechanisms are possible. Some platelets may encounter the surface, fail to adhere, but undergo changes in reactivity as a result of the encounter. Alternatively, prolonged exposure to a substance that inhibits platelet function, such as low concentrations of ADP (lo), may occur as a consequence of surface-protein-platelet adherence of the majority of platelets. Nonadherent platelets in 0.9-m2 circuits probably do not undergo extensive release, since mean platelet PF, content does not decrease to less than 40 pg/lOg platelets during the first 3 h of recirculation. After 3 h, large numbers of detached platelets which have undergone extensive release reenter the circulation, and platelet PF, content decreases below 40 pg/lOg platelets. PF, release probably occurs from alpha granules (23) which are not identical to either lysomal or dense granules, which contain ADP and serotonin. These experiments provide evidence that platelets may exist in extracorporeal perfusion circuits in three different conditions. Platelets may remain unaltered with normal sensitivity to aggregating agents. Platelets may undergo functional changes as suggested by decreased sensitivities in association with minimal changes in mean platelet PF, content during the first 3 h of recirculation in 0.9-m2 circuits. Last, platelets may be irreversibly altered as indicated by low mean platelet PF, contents after 6 h of recirculation in 0.9.m2 circuits. The fact that some platelets remain functionally intact for 6 h of recirculation indicates that under as yet undefined conditions, human platelets can be compatible with extracorporeal surfaces. The Glenn This 19055, tional Received
authors gratefully acknowledge the technical assistance of Chong, Linda Guiod, Alice Kuo, and Maxine Millman. investigation was supported by Grants NOl-HR-42922, HLHL-14217, HL-15226, HL-16583, and HL-18209 from the NaHeart and Lung Institute. for publication
19
July 1976.
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H628
HE~~ESSY
ET AL.
REFERENCES 1. BAIER, R. E., AND R. C. DUTTON. Initial events in interactions of blood with a foreign surface, J, Biomed. Mater. Res. 3: 191-206, 1969. 2. BEAU, A. F. A method for hemoglobin in serum and urine. Am. J. CZin. Pathol, 38: 111-112, 1962. 3. BORN, G. V. R. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature 194: 927-929, 1962. 4. BRECHER, G., AND E. P. CRONKITE. Morphology and enumeration of human blood platelets. J. Appl. Physiol. 3: 365-377, 1950. 5. CARVALHO, A. C. A., R. W. COLMAN, AND R. S. LEES. Platelet function in hyperlipoproteinemia. N. En&. J. Med. 290: 434-438, 1974. 6. DELEVAL, M. R., J. D. HILL, C. H. MIELKE, JR., C. SENITTI, AND F. GERBODE. Platelet kinetics during extracorporeal circulation. Trans. Am. Sot. ArtificiaZ InternaZ Organs 18: 355-357, 1972. 7. DUTTON, R. C., L. H. EDMUNDS, JR., J. C. HUTCHINSON, AND B. B. ROE. Platelet aggregate emboli produced in patients during cardiopulmonary bypass with membrane and bubble oxygenators and blood filters. J. Thoracx Cardiovasc. Surg. 67: 258-265, 1974. 8 FRIEDMAN, L. I., E. F. GRABOWSKI, E. F. LEONARD, AND C. W. MCCORD. Inconsequentiality of surface properties for initial platelet adhesion. Trans. Am. Sot. Artificial InternaZ Organs 16: 63-73, 1970. 9 HAWKEY, C. M., AND C. SYMONS. Variations in ADP-induced platelet aggregation in vitro in primates as a result of differences in plasma ADP inhibitor levels. Thromb. Diath. Haemorrhczg. 19: 29-35, 1968. 10 HOLME, S., AND H. HOLMSEN. ADP-induced refractory state of platelets in vitro. I. Methodological studies on aggregation in platelet rich plasma. Stand. J. HuematoZ. 15: 96-103, 1975. 11 JERUSHALMY, Z., AND M. B. ZUCKER. Some effects of fibrinogen degradation products (FDP) on blood platelets. Thromb. Diczth. Huemorrhug. 15: 413-419, 1966. 12. KIM, S. W., R. G. LEE, H. OSTER, D. COLEMAN, J. D. ANDRADE, D. J. LENTZ, AND D. OLSEN. Platelet adhesion to polymer surfaces. Trans. Am. Sot. Artificial InternaZ Organs 20: 449-455, 1974. 13. KOLOBOW, T., E. W. STOOL, P. K. WEATHERSBY, K. PIERCE, F. HAYANO, AND J. SUAUDEAU. Superior blood compatibility of silicone rubber free silica filler in the membrane lung. Trans.
Am. Sot. ArtificiaZ Internal Organs 20A: 269-277, 1974. 14. LYMAN, D. J., J. L. BRASH, S. W. CHAIKIN, K, G. KLEIN, AND M. CARINI. The effect of chemical structure and surface properties of synthetic polymers on the coagulation of blood. II. Protein and platelet interaction with polymer surfaces. Truns. Am. Sot. Artificial InternaZ Organs 14: 250-255, 1968. 15. MANCINI, G., A. 0. CARBONARO, AND J. F. HEREMANS. Immunochemical quantitation of antigen by single radial immunodiffusion. Immunochemistry 2: 235-254, 1965. 16. MCKENNA, R., F. BACHMANN, B. WHITAKER, J. R. GILSON, AND M. WEINBERG, JR. The hemostatic mechanism after open-heart surgery. J. Thorucic Cardiovasc. Surg. 70: 298-308, 1975. 17. NIEWIAROWSKI, S., C. T. LOWERY, J. HAWIGER, M. MILLMAN, AND S. TIMMONS. Immunoassay of human platelet factor 4 (PF, antiheparin factor) by radial immunodiffusion. J. Lab. CZin. Med. 87: 720-733, 1976. 18. PACKHAM, M. A., G. EVAN, M. G. GLYNN, AND J. F. MUSTARD. The effect of plasma proteins on the interaction of platelets with glass surfaces. J. Lab. CZin. Med. 73: 686-697, 1969. 19. SALZMAN, E. W. Measurement of platelet adhesiveness. J. Lab. CZin. Med. 62: 724-735, 1963. 20. SHATTIL, S. J., R. ANAYA-GALINDO, J. BENNETT, R. W. COLMAN, AND R. A. COOPER. Platelet hypersensitivity induced by cholesterol incorporation. J. CZin. Invest. 55: 636-643, 1975. 21. SINAKOS, Z., AND J. P. CAEN. Platelet aggregation in mammalians (human, rat, rabbit, guinea-pig, horse, dog). A comparative study. Thromb. Diath. Haemorrhag. 17: 99-111, 1967. 22. SNEDECOR, G. W., AND W. G. COCHRAN. StatisticaL Methods. Ames, Iowa: Iowa State Univ. Press, 1967. 23. WALSH, P. N., AND G. GAGNATELLI. Platelet antiheparin activity storage site and release mechanism. BZood 44: 157-168, 1974. 24. WARD, C. A., B. RUEGSEGGER, D. STANGA, AND W. ZINGG. Reduction in platelet adhesion to biomaterials by removal of gas nuclei. Trans. Am. Sot. Artificial InternaZ Organs 20: 77-84, 1974. 25. ZAPOL, W. M., S. BLOOM, A. CARVALHO, T. WONDERS, M. SKOSKIEWICZ, R. SCHNEIDER, AND M. SNIDER. Improved platelet economy using filler-free silicone rubber in long term membrane lung perfusion. Trans. Am. Sot. Artificial Internal Organs 21: 587-592, 1975.
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HENNESSY, VINCENT L., JR., RICHARD E. HICKS, STEFAN NIEWIAROWSKI, L. HENRY EDMUNDS, JR., AND ROBERT W. COLMAN. Function of human platelets during extracorporeal
circuZation. Am. J. Physiol. 232(6): H622-H628, 1977 or Am. J. Physiol.: Heart Circ. Physiol. l(6): H622-H628, 1977. -The interaction between human platelets and nonbiologic surfaces was studied during in vitro recirculation of 500 ml of fresh, heparinized human blood in four different perfusion circuits. Circuits differed in surface area (0.1 m* or 0.9 m*> and in surface composition. No important differences were observed between standard silicone-rubber and filler-free, silicone-rubber surfaces. Platelet counts decreased to 85% of control in 0. lm* circuits, but retained normal sensitivity to aggregating agents and released only small amounts of platelet factor 4 (PF,). In cant rast, platelet counts in 0.9-m* circuits decreased to 20% of control within 2 min and platelet sensitivity was depressed out of proportion to the fall in platelet count. Plasma PF, progressively increased and platelet PF, content progressively decreased during 6 h of recirculation. The results indicate that human platelets may exist in three conditions during extracorporeal circulation. Some platelets are unaltered, some are less sensitive to aggregating agents, and others have undergone extensive release. The ratio of blood volume to surface area appears to be an important determinant of platelet-surface interaction.
19104, and University
study the effects of both surface composition (SSR and FFSR) and surface area on human platelets, and to distinguish reversible and irreversible changes in platelets during extracorporeal perfusion. METHODS
Perfusion circuits. Perfusion circuits with surface areas of 0.1 m2 or 0.95 m2 were assembled from siliconerubber tubing (3/8 in ID, 42 in long, Dow Corning Corp., Midland, Mich. >, silicone-rubber venous reservoirs and spiral-coil oxygenators (Sci-Med Life Systems, Inc., Minneapolis) (Fig. 1). Surface areas were calculated from measured dimensions of the reservoir, tubing, and oxygenator blood contact surfaces. Half of the SSR components were coated and cross-linked with FFSR, Circuits differed by the presence or absence of the spiralcoil-membrane oxygenator (surface areas: membrane, 0.8 m2; cylinder-blood contact surface, 0.05 m2) and by the presence or absence of the FFSR coating. Except for polycarbonate connectors, which represented 3.8 and 0.40% of the surface areas of the O.l-m2 and 0.9-m2 circuits, respectively, all blood contact surfaces were either SSR or FFSR. Smaller circuits of SSR and FFSR filler-free silicone rubber; platelet-surface interaction; platelet were designated 1s and IF, respectively; larger circuits factor 4; platelet aggregation of SSR and FFSR were designated 9s and 9F, respectively. Sci-Med Life Systems, Inc. provided sterile standard WHEN BLOOD CONTACTS a nonbiologic surface, plasma silicone-rubber oxygenators and oxygenators coated and cross-linked with FFSR. Other silicone-rubber compoproteins are adsorbed and platelets adhere (1, 14, 18). During extracorporeal perfusion in the presence of hep- nents for FFSR circuits were filled with a 7% dispersion arin the number of circulating platelets decreases 40- of FFSR in toluene (Sci-Med Life Systems, Inc.). After 60% and the function of remaining platelets is depressed drainage and evaporation of toluene at 23”C, tubing and reservoirs were filled with 100% nitrogen and were irra(19). Deficiency in the number and function of platelets has been suggested as an important cause of bleeding diated at 23°C with 5-10 megarads from a cobalt-60 source (U.S. Army Natick Laboratories, Natick, Mass.). following open-heart operations (16). Efforts to produce a totally nonthrombogenic surface All components were sterilized in ethylene oxide and have not been successful. Recently, Kolobow et al. (13) degassed for at least 72 h before the circuits were assemand Zapol et al. (25) observed that silica particles used bled. Circuit priming. Blood, 500 ml, was drawn from fastas filler to increase the tensile strength of commercial ing donors through siliconized needles and polyvinyl silicone rubber contributes to the loss of platelets during partial cardiopulmonary bypass in lambs. By coating tubing directly into the venous reservoir, which constandard silicone rubber (SSR) of extracorporeal perfu- tained 2,500 U of beef-lung heparin (The Upjohn Comsion systems with filler-free silicone rubber (FFSR) pany, Kalamazoo, Mich.) and 1.650 mg glucose. All which lacks silica particles, they reduced the loss of donors had abstained from all medication for at least 2 platelets. The present experiments were designed to wk prior to their giving blood. H 622
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PLATELET
FUNCTION
DURING
EXTRACORPOREAL
0.1 M2 FIG.
1. Diagrams
0.9 of 0. l-m2
and 0.9-m’
recirculation
CIRCULATION
M2 circuits.
Blood and gas compartments of the 0.9-m2 circuits were flushed with filtered ‘100% carbon dioxide for at least 30 min before priming. Oxygenators were primed by applying a vacuum to the lower gas port and permitting blood to enter the device by gravity. Tubing was filled by gravity. After priming, no gas bubbles were visible in any circuits when recirculation was started. Recirculation system. Blood was recirculated by a precisely shimmed, barely occlusive, calibrated doubleroller pump (Cardiovascular Instrument Corp., Wakefield, Mass.) at a rate equal to twice the remaining circuit blood volume per minute. An adjustable screw clamp maintained circuit back-pressure at 300 mmHg. Blood temperature was maintained at 37°C by immersion of the venous reservoir in a water bath. A mixture of 95% oxygen and 5% carbon dioxide was passed through the gas compartment of the oxygenator at 1 literlmin, countercurrent to the direction of blood flow. Blood was recirculated for 6 h in each circuit on three occasions, for a total of 12 trials. Control samples for platelet studies were obtained directly from the donor (45 ml of blood in 250 U heparin and 165 mg glucose), and were studied immediately and after 1, 3, and 6 h of incubation at 37°C. Samples were taken from the circuit after 2 min of recirculation and after 1, 3, and 6 h of recirculation. Additional samples obtained after 2 min of recirculation were studied after 1, 3, and 6 h of incubation at 37°C. Further samples for platelet counts only were obtained after 15 and 30 min and at 2,4, and 5 h of recirculation. Plasma hemoglobin was measured after 6 h of recirculation in each circuit (2). PlateLet studies. Platelet-rich plasma (PRP) was prepared in plastic ware at 23°C by adding 1 vol of 3.8% sodium citrate to 9 vol of heparinized blood, centrifuging the mixture for 10 min at 170 xg, and gently aspirating the PRP. The platelet count was 300-400,000, unless otherwise specified. The remaining blood was centrifuged for 4 min at 12,000 xg in an Eppendorfcentrifuge
H623 (Brinkmann Instruments, Inc., Westbury, N.Y.) to obtain platelet-poor plasma (PPP), which contained fewer than 1,000 platelets per microliter. Platelets were counted by phase microscopy (4). Platelet aggregation was studied in an aggregometer attached to a strip-chart recorder (Chrono-Log Corp., Havertown, Pa.) with use of a previously described modification (5) of the method of Born (3). PRP was adjusted to 10% light transmission and PPP to 90% light transmission. The extent of aggregation was determined by the maximum percent difference between PRP and PPP achieved in 10 min. As determined previously in 15 normal individuals, complete second-wave aggregation can be assumed (at a 95% confidence level) when PRP light transmittance is greater than 61% for ADP, greater than 57% for epinephrine (unpublished data), and greater than 70% for collagen (5). Because of the apparent log-normal distribution of platelet sensitivities in control subjects, sensitivities were recorded as the log of the concentration of dose of the aggregating agent (5). A second criterion of the second wave of aggregation is the amount of serotonin released from PRP as measured by the method of Jerushalmy and Zucker (11). Normal PRP was incubated with 0.01 vol 14C-labeled serotonin ( [14C]5-hydroxytryptamine, 30 &i/ml; New England Nuclear, Boston). Release was produced by adding respective aggregating agents to labeled PRP stirred at 1,200 rpm at 37°C for 15 min. After centrifugation at 12,000 xg for 4 min, the percentage of 14C-labeled serotonin in the supernatant was expressed as a percentage of labeled serotonin originally taken up. Greater than 20% for ADP, greater than 25% for epinephrine (unpublished data), and greater than 40% for collagen (20) indicated complete second-wave aggregation at a confidence level of 95%. The sensitivity of threshold of platelets to aggregation was defined as the lowest concentration of aggregating agents that produced a complete second wave of aggregation, as judged by aggregometry and 14C-labeled serotonin release. A correlation coefficient of 0.95 between serotonin release and full aggregation was previously demonstrated (20) and was observed in these experiments. Predicted platelet sensitivity curves were constructed for each aggregating agent at low platelet counts by determining platelet sensitivities of PRP from 14 donors which was diluted with the donors’ own respective PPP to platelet counts of 100,000/mm3 and less. In 0.9-m2 circuits, in which immediate thrombocytopenia occurred, a sample of unexposed donor blood was diluted with its own PPP to an equally low platelet count for control platelet function studies. The maximum aggregating doses, even if aggregation failed to occur, were considered to be 50 PM of ADP, 50 ,ug/ml of acid-soluble collagen (5.5 mg/ml; Worthington Biochemical Corp.), and 250 PM epinephrine. Platelet factor 4 (PF,) was measured by the modified radial immunodiffusion method of Mancini et al. (15) with a monospecific antibody against purified PF4, as described by Niewiarowski et al. (17). PF, in plasma was measured after release and centrifugation, as de-
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H624
HENNESSY
scribed for 14C-labeled serotonin. PF, in platelets was measured by determining the total concentration in frozen PRP after exposure to 0.8% Triton X-100 and subtraction of the PF, in PPP. Platelet PF, was expressed in micrograms per log platelets. Scanning electron microscopy. Scanning electron microscopy was used to study differences between SSR and FFSR surfaces before blood contact and after 6 h of recirculation. The perfusion circuit was rinsed with 1 liter Ringer-lactate which contained 1,000 U of beeflung heparin (Upjohn). Random samples of reservoir, tubing, and oxygenator membrane were excised and placed in buffered 3% glutaraldehyde. After fixation, samples were rinsed in distilled water and dehydrated by immersion in increasing concentrations of acetone. Specimens were transferred into liquid carbon dioxide from absolute acetone to which 1 ml of amyl acetate had been added to identify residual organic solvent. Criticalpoint drying was done using liquid CO, in a Denton critical-point dryer (Denton Vacuum Inc., Cherry Hill, N. J. ). Scanning electron microscopy was performed with use of a model JSM 50-A scanning electron microscope from Electron Optic Laboratories Ltd., Tokyo, to obtain qualitative information on adherent platelets. StatisticaL analysis. Observations at each sampling time were considered to be independent of observations at other sampling times to simplify statistical analysis. At each sampling time, mean platelet counts, mean platelet sensitivities, platelet PF, concentrations, plasma PF, concentrations, and plasma hemoglobin concentrations were compared between the four circuits by two-way analysis of variance (22). Since, with one exception, no differences between FFSR and SSR circuits were found, data from FFSR and SSR circuits were combined to compare data from 0. l-m2 and 0.9-m* circuits. The Student paired-t test was used to compare observations within circuits at different sampling times (22) . RESULTS
PZasma hemoglobin. Surface composition did not alter plasma hemoglobin significantly (P > 0.05) after 6 h of recirculation in the 0. l-m2 circuits (Fig. 2). However,
ET AL.
plasma hemoglobin increased significantly when surface area was increased from 0.1 m2 to 0.9 m2 (P < 0.01). PLatelet number. Mean changes in platelet count, expressed as a percentage of the platelet count of blood drawn directly from the donor, are plotted in Fig. 3. In both O.l-m2 circuits, a 10% decrease in platelet count, which was significant (P < 0.05), occurred after 2 min of recirculation. Subsequent platelet counts within circuits remained significantly decreased (P < 0.05) from control counts, but did not differ significantly (P > 0.05) from each other. Platelet counts from 1s circuits did not differ significantly (P > 0.05) from counts from 1F circuits at any sampling time. Platelet counts in both 0.9-m2 circuits fell within 2 min of recirculation to 20% of control (P < 0.01). Over the next 6 h, platelet numbers rebounded to 55% and 41% of control levels in 9s and 9F circuits, respectively. Although the platelet counts in 9s and 9F circuits were not significantly different from each other at any sampling time (P > 0.05), counts at 6 h represented a significant increase from counts after 2 min of recirculation (P < 0.01). Platelet counts from O.l-m2 circuits differed significantly (P < 0.01) from those of 0.9-m2 circuits at all sampling times except control. PLateLet sensitivity. The response of platelets to ADP and epinephrine for each circuit is presented in Fig. 4. In general, data from collagen studies paralleled those from ADP and epinephrine but had wider ranges. Platelet sensitivity to the three aggregating agents did not differ significantly between 1s and 1F circuits at any sampling time. A significant decrease in platelet sensitivity to ADP and collagen occurred after 2 min of recirculation in 0. l-m2 circuits (P < 0.02); however, the initial decrease in platelet sensitivity to epinephrine was not significant (P > 0.05). A more gradual, but significant (P < 0.05), decrease in platelet sensitivity to all three aggregating agents occurred over the next 6 h in recirculated platelets in both 1s and 1F circuits and in platelets of incubated donor blood. However, the changes in platelet sensitivity during recirculation did not differ significantly (P > 0.05) from changes in sensi% of Control Platelet Count
Hb mg/lOOml 250200
-
l50I
1
1
I
i
I
2
3 (hours)
lime
1
4
1
5
J
6
3. Mean changes in platelet count during recirculation in each circuit. Platelet counts are plotted as a percentage of the platelet count of blood drawn directly from donors. o-o, O.l-m2 SSR circuit; o----- o, O.l-m2 FFSR circuit; o-o, 0.9-m2 circuit; l -----0, 0.9-m2 FFSR circuit. Each point is mean of 3 platelet counts. Point i indicates 2 min of recirculation in this and subsequent figures. FIG.
IS FIG.
deviations
2.
Mean after
plasma hemoglobin 6 h of recirculation
IF
9s
9F
concentrations i n each perfusion
with standard circuit.
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PLATELET ADP llM
t
FUNCTION
DURING
EXTRACORPOREAL
FFSR
SSR Cwcuits
1
L
I
I
1
I
L
1
I
3 4 (hours)
5
6
CIRCULATION
Orcults
Epmephrlne 9M
2 Time
I
I
i
I
I
I
I
I
I
23456 Time (hours)
FIG. 4. Lowest mean concentration of ADP and epinephrine required to produce a complete 2nd wave of aggregation as determined by light transmission or serotonin release is presented for blood recirculated in each circuit and for simultaneous control studies. Data from standard silicone-rubber (SSR) circuits appear in left column of graphs, and data from filler-free, silicone-rubber (FFSR) circuits are in right column. oo indicates blood recirculated in 0. l-m2 circuits. l l indicates blood recirculated in 0.9-m2 circuits. clq indicates donor blood incubated at 37°C for 6 h. X-X indicates expected platelet sensitivities for thrombocytopenic samples ( 0.05). Although platelet numbers increased after 6 h of recirculation in 0.9-m* circuits, platelet sensitivity did not improve.
Surface area as opposed to surface composition appeared to be the critical factor in the significant decrease (P < 0.05) in platelet sensitivity to ADP and epinephrine at all sampling times when 0.1-m* and 0.9m2 circuits were compared. Platelet sensitivity to collagen was significantly depressed (P < 0.02) on initial exposure to both 0.1-m* and 0.9-m* circuits; but thereafter, no significant differences (P > 0.05) in platelet sensitivity to collagen occurred as a function of surface area, surface composition, or duration of recirculation. The possibility that observed changes in platelet sensitivity between 0. l-m2 and 0.9-m* circuits may have been due to greater concentrations of ADP or other substances released from hemolyzed red cells was studied. Plasma hemoglobin concentrations between 50 and 300 mg/lOO ml were produced by forcing blood through a no. 25 needle. The blood was then centrifuged at 12,000 x g for 4 min and the hemolyzed plasma obtained was added to PRP prepared from freshly donated blood. Platelet sensitivities to all three aggregating agents were not altered by the addition of this hemolyzed plasma with hemoglobin concentrations up to 300 mg/ 100 ml. The pH in 1s and 1F circuits ranged from 7.36 to 7.41, while that in 9s and 9F circuits ranged from 7.23 to 7.33. To rule out the possibility that changes in pH might affect platelet sensitivity to aggregating agents, an additional control experiment was carried out. The pH of platelet-rich plasma prepared from each of two donors was adjusted to values between 7.12 and 7.37 by bubbled 100% carbon dioxide; the platelet sensitivities to ADP, epinephrine, and collagen were then measured and compared to sensitivities of control aliquots of plateletrich plasma from the same donors. Changes in pH between 7.12 and 7.37 did not affect platelet sensitivities to ADP, epinephrine, and collagen. Similarly, the possibility that increased blood oxygen tension might affect platelet sensitivities possibly by producing reactive intermediates was tested by recirculating fresh donor blood in a 0.9-m* circuit in which the oxygenator was ventilated with 90% nitrous oxide and 10% oxygen. Complete second-wave aggregation as measured by 14C-labeled serotonin release occurred at 47.4 PM t 13.9 for ADP and 54.8 PM t 14.6 for epinephrine (from 2 min to 6 h), and the plotted curves were similar to those for 0.9-m* circuits in Fig. 4. Platelet factor 4 in platelets and plasma. Plasma PF, antigen was less than 0.5 pg/ml plasma in control samples, which is the lower limit of the sensitivity of the method (17). PF, antigen was detected in all circuits after 2 min of recirculation (P < 0.01) (Fig. 5). The increase in both FFSR circuits at 2 min over control samples of unrecirculated blood was less than the increase in SSR circuits, and these differences were significant (P < 0.05). However, after 2 min surface composition did not significantly alter plasma PF,. Plasma PF, increased more rapidly in 0.9-m* circuits and was significantly (P < 0.01) greater than in 0.1-m* circuits at each sampling time after 2 min. Platelet PF, did not change significantly (P > 0.1) in any circuit during the first 3 h of recirculation (Fig. 6). The apparent increase at 2 min in 9F circuits is unex-
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H626
HENNESSY
plained, but is not significant. After 6 h of recirculation, platelets in 0.9-m2 circuits lost most of their PF, content (P < 0.02) and differed significantly (P < 0.01) from PF, contents of platelets recirculated in 0. l-m2 circuits. Surface composition did not significantly (I’ > 0.05) alter PF, content of platelets at any sampling time. Platelet factor 4 values were similar in the 0.9-m2 circuit in which the oxygenator was ventilated with 90% nitrous oxide and 10% oxygen. Under these conditions mean platelet PF, content decreased progressively from 76.5 pg/lOs platelets at 2 min to 30.9 pg/lOs platelets at 6 h. Plasma PF, rose from 4.6 pg/ml at 2 min to 11.7 pug/ ml at 6 h. Scanning electron microscopy. Striking differences in SSR and FFSR surfaces that were not exposed to blood
ET AL.
were not observed, although several photographs suggested that FFSR surfaces were smoother and did not contain protruding silica particles. Qualitative differences in the adhesion of blood elements to SSR and FFSR surfaces were not observed in the random samples of circuit components studied after 6 h of recirculation (Fig. 7). Both single and clumps of platelets and swollen, deformed red cells adhered to both surfaces. Only a few fibrin strands were observed. DISCUSSION
The in vitro recirculation model controls many variables which may affect platelet function during extracorporeal circulation. Fresh heparinized human blood obPF4 J.lg/PltxlO* 200 r 160 I t I ‘1
PF4
ug/ml 24i-
2 3 4 Time (hours)
5
6
Time
(hours)
FIG. 5. Mean changes in plasma platelet factor 4 content during recirculation in each circuit. Symbols are the same as in Fig. 3. Initial values from blood drawn directly from donor were less than 0.5 @g/ml.
FIG. 6. Mean changes in platelet factor 4 content during recirculation in each circuit. Symbols are the same as in Fig. 3. Initial values indicate platelet factor 4 content in blood drawn directly from donors.
FIG. 7. Representative scanning electron micrographs of an SSR oxygenator membrane (A) and an FFSR oxygenator membrane (B) after 6 h of recirculation (x300). P indicates a single platelet; PP indicates a group of platelets. RBC indicates adherent red cells. F
indicates fibrin strands. At high magnification, oxygenator membranes are irregular. No morphologic differences in number and shape of platelets between SSR and FFSR surfaces were seen at magnifications between 100 and 10,000.
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PLATELET
FUNCTION
DURING
EXTRACORPOREAL
H627
CIRCULATION
viates potential extrapolative problems involved in the use of animal platelets. Since animal and human platelets differ in response to aggregating agents (9,21), they may differ in response to foreign surfaces. Absence of an animal in the circuit prevents catabolism of released substances, clearance or sequestration of altered platelets (6), and addition of newly produced platelets to the system. Changes in number and function of platelets and platelet composition can be quantitated. Finally, because platelets are minimally altered in 0.1.m2 circuits, new surface materials can be easily studied by inseries addition to this basic test circuit. The data clearly show that filler-free silicone rubber offers no advantage over standard silicone rubber in the preservation of human platelets during extracorporeal circulation. With the exception of slight differences in plasma PF, after 2 min of recirculation, no differences in platelet numbers, function, or release reaction were observed between the two types of surfaces studied. We conclude that the observed differences noted during partial cardiopulmonary bypass in lambs by Kolobow et al. (13) and Zapol et al. (25) may be related to species differences in platelets or inclusion of animals in the circuit, rather than to surface composition of the bypass circuit. Large numbers of plateletsadhere to the 0.9-m2 surface with first contact. The decrease in platelet count is much greater than that observed in 0.1.m2 circuits. Procedures used in priming oxygenators reduce the likelihood that decreased platelet counts in 0.9-m2 circuits are due to microbubbles (24). Under the same conditions of blood volume and flow rate, the decrease in platelet count appears directly proportional to surface area. Although many potential factors may govern platelet-surface interaction, only flow rate, duration of exposure, and the composition of adsorbed proteins are known to influence platelet adhesion to nonbiologic surfaces (1, 8, 12, 14, 18). In the 0. l-m2 circuits, the ratio of blood to surface area is 1 ml/2 cm2. In 0.95m2 circuits, the ratio is 1 ml/19 cm2. The ratio of an individual’s blood volume (excluding priming fluids) to the surface area of most membrane oxygenator systems is approximately 1 ml/lo-12 cm2. This ratio is consistent with observed changes in platelet number and function during short-term (l-2 h) total cardiopulmonary bypass in humans (7, 16). Consequently, more efficient oxygenators with a reduced surface area may mitigate changes in platelets caused by extracorporeal circulation. Platelet count rose progressively after 1 h of recirculation in 0.9-m2 circuits. This observation clearly indicates that some initially adherent platelets become detached. The progressive rise in platelets correlates with a progressive rise in plasma PF, content and a decrease in mean platelet PF, content to less than 40 pg/lOg platelets. These data indicate that detached platelets have undergone extensive release and are incapable of secondary aggregation and release. Most changes in platelet number and function occur
within 2 min of recirculation. In 0.1.m2 circuits, a 15% decrease in platelet count and a modest increase in plasma PF, occur. Further changes in platelet count, platelet function, and PF, content and release do not occur during the subsequent 6 h. The data do not exclude the possibility that adherent platelets may become detached and may not function, since the experimental error of platelet counts and sensitivity of aggregometry cannot detect the admixture of up to 7% of detached nonfunctioning platelets. The important observation is that the remaining nonadherent platelets function normally and do not react with extracorporeal surfaces. Blood contact surfaces of 0.1-m’ circuits become compatible with platelets and are “inactivated” by contact with 500 ml of fresh, heparinized blood. This observation suggests that platelet-compatible nonbiologic surfaces can be developed. The immediate change in function of nonadherent platelets in 0.9-m2 circuits contrasts with the apparently normal function of the majority of circulating platelets in 0.1.m2 circuits. In 0.9-m2 circuits loss of sensitivity of nonadherent platelets within 2 min is unexplained, but at least two mechanisms are possible. Some platelets may encounter the surface, fail to adhere, but undergo changes in reactivity as a result of the encounter. Alternatively, prolonged exposure to a substance that inhibits platelet function, such as low concentrations of ADP (lo), may occur as a consequence of surface-protein-platelet adherence of the majority of platelets. Nonadherent platelets in 0.9-m2 circuits probably do not undergo extensive release, since mean platelet PF, content does not decrease to less than 40 pg/lOg platelets during the first 3 h of recirculation. After 3 h, large numbers of detached platelets which have undergone extensive release reenter the circulation, and platelet PF, content decreases below 40 pg/lOg platelets. PF, release probably occurs from alpha granules (23) which are not identical to either lysomal or dense granules, which contain ADP and serotonin. These experiments provide evidence that platelets may exist in extracorporeal perfusion circuits in three different conditions. Platelets may remain unaltered with normal sensitivity to aggregating agents. Platelets may undergo functional changes as suggested by decreased sensitivities in association with minimal changes in mean platelet PF, content during the first 3 h of recirculation in 0.9-m2 circuits. Last, platelets may be irreversibly altered as indicated by low mean platelet PF, contents after 6 h of recirculation in 0.9.m2 circuits. The fact that some platelets remain functionally intact for 6 h of recirculation indicates that under as yet undefined conditions, human platelets can be compatible with extracorporeal surfaces. The Glenn This 19055, tional Received
authors gratefully acknowledge the technical assistance of Chong, Linda Guiod, Alice Kuo, and Maxine Millman. investigation was supported by Grants NOl-HR-42922, HLHL-14217, HL-15226, HL-16583, and HL-18209 from the NaHeart and Lung Institute. for publication
19
July 1976.
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