Storage and transfusion of platelets collected by an automated two-stage apheresis procedure T. SIMON, E.J. LEE, A. HEATON,C.A. SCHIFFER, D. HUESTIS,D. SCHOENDORFER, S. HEDBERG, AND D. BUCHHOLZ Platelets collected by using a two-stage automated blood cell separator were evaluated after 5 days of storage. The procedure caused no unanticipated physiologic changes in donors and produced > 3 x 10’’ intact platelets in 200 mL of plasma, plus an additional 400 mL of plasma with intact coagulation factors. Posttransfusion recovery of autologous radiolabeledplatelets was comparable to that seen in platelets prepared by manual centrifugation techniques. Corrected count increments in 14 patients showed results similar to those with control transfusions. This device, which involves a collection time of less than 90 minutes, provides an option for plateletpheresis in a variety of settings including blood mobiles. TRANSFUSION 1992;32:624628.
Abbrevlatlons: CCl(s) = corrected count Increment(s); PC(s) = platelet concentrate(8); PRP = platelet-rich plasma.
gram of the disposable set composed of polyvinylchloride tubing and bags for plasma and PRP, rigid molded plastic (acrylic) parts, anticoagulant (ACD-A), and PC storage containers (PL732 plastic, Fenwal). Blood is removed from the donor at rates of up to 100 mL per minute, and red cells are transfused at 130 mL per minute, by using a single venous access. A sophisticated pressure and flow monitoring system optimizes the flow rates for each donor. Recommended anticoagulant ratios are 6 to 8 percent. Blood from the donor is initially directed.to one side of a dual-chambered reservoir (100-mL capacity). Blood is pumped from the reservoir to the Plateletcell separator at approximately one-half of the donor’s blood withdrawal rate (50 mumin). PRP is sent to a collection reservoir, while red cells are pumped from the separator to the other side of the reservoir and then returned to the donor when the reservoir becomes full (about 100 mL). Meanwhile, the reserve of whole blood in the other side of the reservoir is used to supply the separator continuously while red cells are being returned. Thus, the system processes the donor’s blood without interruption and continuously provides a flow of PRP (about 10 mumin). Blood processing continues until the desired weight of PRP is attained. A detector measures the optical characteristics of the PRP output of the Plateletcell separator and modifies the rate of PRP flow and the revolutions per minute (rpm) of the separator to produce maximum light absorption (i.e., maximum platelet concentration) with minimal red cell and/or white cell contamination. If such contamination exceeds predetermined thresholds, the instrument diverts that PRP back into the reservoir for repeat processing. Figure 2 shows a cutaway diagram of the Plateletcell separator. Centrifugal force is used to separate the blood components in the separator. The rotor contains a magnetic drive element that provides a rotational energy coupling it and the instrument. The instrument spins the rotor at speeds up to 3600 rpm inside the stationary case. As there are no external mechanical seals, the Plateletcell separator, like the separation set (Plasmacell-C, Fenwal), is a biologically closed system.
SEVERAL APHERESIS SYSTEMS allow the collection of 3 to 4 x 10l1platelets in a closed or functionally closed system that permits extended The Autopheresis-C instrument (Fenwal Automated Systems, Santa Ana, CA), originally developed for plasma collection, can be used with a disposable blood processing set (Plateletcell, Fenwal) to prepare single-donor platelet concentrate (PC). In the first step, 600 to 700 mL of plateletrich plasma (PRP) is collected from donors by using a single venous access and a separation process with an extracorporeal volume of less than 100 mL. In a second, donor-independent step, platelets are concentrated to a volume of approximately 200 mL by using a spinning membrane plasma separation device integrally attached to the processing unit. This system w a s evaluated in four laboratories; results show that platelets can be harvested successfully with the Autopheresis-C system.
Materials and Methods The platelet collection method using the Autopheresis-C system has been described previ~usly.~ Figure 1 shows a diaFrom the Transfusion Medicine Center, University of New Mexico School of Medicine, and United Blood Services, Albuquerque, New Mexico; the University of Maryland Cancer Center and the Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland; the Mid Atlantic Region American Red Cross Blood Services, Norfolk, Virginia; the Hemotherapy Service, University of Arizona Health Sciences Center, Tucson, Arizona; and Fenwal Division, Baxter Healthcare Corporation, Deerfield, Illinois. Supported by grants from Fenwal Division, Baxter Healthcare Corporation. Some of the studies were conducted in the General Clinical Research Center, University of New Mexico, DRR NIHJ-MOIORR00997-15. Rcceived for publication September 24,1991; revision received January 13, 1992, and accepted January 24, 1992.
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TRANSFUSION 1992-Vol. 32. No. 7
AUTOMATED TWO-STAGE PIATELETPHERESIS
625
is exposed to centrifugal forces as high as 300 x g (depending on the rpm). The PRP, being the less dense component, accumulates toward the center of rotation, where it exits the separation area through a series of ports that lead to the PRP outlet at the bottom of the case. Meanwhile, “packed cells” are separated from the center of the rotation where they exit the rotor through a series of packed cell ports. An integrally connected Plasmacell-C separator is provided with the kit for use in concentration of PRP. Once the desired volume of PRP is harvested, the instrument is disconnected from the donor, the separation device is discarded, and the Plasmacell-C device and tubing set are installed on the instrument. The collected PRP is concentrated to approximately 200 mL of PC; 400 to 500 mL of platelet-poor plasma is also collected as a byproduct.
In vitro studies
LJ
FIG. 1. The Plateletcell disposable set. Components on the left side of the seal are used to collect platelet-rich plasma. To the right is the platelet concentration set, installed after the donor is disconnected. PC = platelet concentrate; PPP = platelet-poor plasma; PPC = primary platelet concentrates. Reprinted with permission from Vox Sang 1990;58:100-5.
Outlet
Eleven healthy volunteers (6 men, 5 women) meeting all accepted standards for blood donors (United Blood SeMces, American Red Cross, American Association of Blood Banks, and U.S. Food and Drug Administration) participated in plateletpheresis for in vitro evaluation of collected cells. Donor age and weight ranged from 24 to 65 years and 138 to 200 pounds, respectively. Donor blood counts were performed with an automated cell counter (Coulter Electronics, Hialeah, FL) and platelet morphology score was analyzed by the methods of Kunicki5 and platelet hypotonic shock by previously described mcth0ds.l Mean platelet volume diameter and size dispersion were measured on a counter (SPlus, Coulter Electronics); donor fibrinogen, prothrombin time, and partial thromboplastin time measurements were measured by standard technique^;^ and total protein was measured by colorimetric biuret reaction (Ektachem, Eastman Kodak, Rochester, NY).The C3a component of complement was assessed by a radioimmunoassay (Amersham, Arlington Heights, IL). Protein electrophoresis as well as prothrombin time and activated partial thromboplastin time were performed on collected platelet-poor plasma. Coagulation factors V, VIII, IX, and X were measured by previously described technique^.^ Red cell and white cell counts in platelet concentrates were performed manually. Platelet aggregation testing of collected platelets used standard techniques with sinmoVL] and gle agonists (ADP at 1.8 x M [1.8 x 4.5 x 10-6M [4.5 x 10-6 mol/L], collagen at 0.17 mg/dL, and epinephrine at 0.9 x lo-‘ M [0.9 x lo-‘ moVL]) and double agonists (collagen and epinephrine at 0.15 mg/dL and 0.8 x M [0.8 x moVL], respectively, and ADP M [0.8 x and epinephrine at 1.6 and 0.8 x moV L], respectively). The extent or intensity of the aggregation reaction (total extent of primary plus secondary responses to the agonists) was expressed as a percentage of the maximum transmittance seen with the platelet-poor plasma. The slope of the reaction in degrees from the horizontal was measured along with, and the lag time from the time of addition of the agonist to the time at which aggregation started. An aggregometer (Pap 4, Biodata Corp., Hatboro, PA) was utilized. Samples were tested within 2 to 3 hours of collection.
Ro. 2. Plateletcell separator, shown diagrammatically by cutting away the surface. PRP = platelet-rich plasma. Reprinted with permission from Vox Sang 1990;58:10&5.
Anticoagulated whole blood enters the lower port of the Plateletcell device and is directed into the bottom of the rotor through a series of inlet slots. Once inside the rotor, the blood
In vivo viability studies in normal volunteers Normal volunteers meeting the same standards as volunteers for the in vitro studies participated. Paired autologous platelet recovery and survival studies were performed in five female and six male subjects aged 20 to 40. On one occasion, standard PC prepared from whole blood was studied after 5 days’ storage in a PL-732 bag; on the other occasion, platelets collected
626
with the Autopheresis-C were studied after 5 days’ storage. Radiolabeled transfusion studies were separated by a minimum of 3 weeks. One laboratory used T r radiolabeling and one used lllIndium, according to published standard methodology.s We analyzed results by both the linear and the computer-assisted gamma function methods.
In vivo platelet transjkions in patients Platelets prepared with the device were stored for up to 5 days and then provided to thrombocytopenic patients requiring transfusion. Whenever possible, we also provided pooled random-donor platelets to each patient before the test transfusion to compare the patients’ response to the test platelets (12 patients). The control transfusion was the next transfusion for patients who had received no platelet transfusion before the test transfusion. Corrected count increments (CCIs) were calculated at 0 to 2 hours (in 2 cases, at 6 hours) and 18 to 24 hours after transfusion by the following formula: CCI =
TRANSFUSION
SIMON ET AL.
Measured rise in platelet count x body surface area platelets transfused ( x loll)
Platelet counts were measured by standard techniques. We studied patients who required platelet transfusions for their clinical management; those who were clinically or serologically alloimmunized or who had splenomegaly or disseminated intravascular coagulation were excluded from the study. In all of the above studies, all donors and patients gave informed consent according to the requirements of the institutional review boards at the study locations.
Results
In vitro studies One of 11 donors had an untoward reaction, consisting of a feeling of warmth and lightheadedness with a slight drop in pulse rate without a change in blood pressure. This disappeared in 5 minutes. Donor hematologic data are shown in Table 1. There were no unanticipated changes after donation. An average of 650 mL of PRP was collected (mean, 3.85 f 0.6 x 10” platelet yield). Following concentration, the PCs had an average volume of 197 mL and contained a mean of 3.68 x 10” platelets. The platelet recovery from PRP ranged from 88 to 101 percent. Morphology scores averaged 295, as compared to 303 before concentration (maximum, 400). Hypotonic shock response did not change significantly after concentration. An average of 6.1 Table 1.
Predonation and postdonation laboratory rests in normal donors (n = 1 7 ) (mean t 1 SD)
Hematocrit (“YO) White cells x 103/pL Platelet count x 103/kL* Prothrombin time (sec) APTTt (sec) Fibrinogen (mg/dL) Total protein (gldl) C3a (ng/mL)
Predonation results
Postdonation results
46 6.0 254 12.7 37 324 6.1 258
47 6.9 180 12.8 36 295 5.6 283
f3
f 1.7
47 0.4 f4 f 63 f 0.6 2 118 f 2
f3 f 1.8
32 2 0.4 f4 f 60 f 0.4 2 131
f
* Difference between predonation and postdonation values: p
Abbrevlatlons: CCl(s) = corrected count Increment(s); PC(s) = platelet concentrate(8); PRP = platelet-rich plasma.
gram of the disposable set composed of polyvinylchloride tubing and bags for plasma and PRP, rigid molded plastic (acrylic) parts, anticoagulant (ACD-A), and PC storage containers (PL732 plastic, Fenwal). Blood is removed from the donor at rates of up to 100 mL per minute, and red cells are transfused at 130 mL per minute, by using a single venous access. A sophisticated pressure and flow monitoring system optimizes the flow rates for each donor. Recommended anticoagulant ratios are 6 to 8 percent. Blood from the donor is initially directed.to one side of a dual-chambered reservoir (100-mL capacity). Blood is pumped from the reservoir to the Plateletcell separator at approximately one-half of the donor’s blood withdrawal rate (50 mumin). PRP is sent to a collection reservoir, while red cells are pumped from the separator to the other side of the reservoir and then returned to the donor when the reservoir becomes full (about 100 mL). Meanwhile, the reserve of whole blood in the other side of the reservoir is used to supply the separator continuously while red cells are being returned. Thus, the system processes the donor’s blood without interruption and continuously provides a flow of PRP (about 10 mumin). Blood processing continues until the desired weight of PRP is attained. A detector measures the optical characteristics of the PRP output of the Plateletcell separator and modifies the rate of PRP flow and the revolutions per minute (rpm) of the separator to produce maximum light absorption (i.e., maximum platelet concentration) with minimal red cell and/or white cell contamination. If such contamination exceeds predetermined thresholds, the instrument diverts that PRP back into the reservoir for repeat processing. Figure 2 shows a cutaway diagram of the Plateletcell separator. Centrifugal force is used to separate the blood components in the separator. The rotor contains a magnetic drive element that provides a rotational energy coupling it and the instrument. The instrument spins the rotor at speeds up to 3600 rpm inside the stationary case. As there are no external mechanical seals, the Plateletcell separator, like the separation set (Plasmacell-C, Fenwal), is a biologically closed system.
SEVERAL APHERESIS SYSTEMS allow the collection of 3 to 4 x 10l1platelets in a closed or functionally closed system that permits extended The Autopheresis-C instrument (Fenwal Automated Systems, Santa Ana, CA), originally developed for plasma collection, can be used with a disposable blood processing set (Plateletcell, Fenwal) to prepare single-donor platelet concentrate (PC). In the first step, 600 to 700 mL of plateletrich plasma (PRP) is collected from donors by using a single venous access and a separation process with an extracorporeal volume of less than 100 mL. In a second, donor-independent step, platelets are concentrated to a volume of approximately 200 mL by using a spinning membrane plasma separation device integrally attached to the processing unit. This system w a s evaluated in four laboratories; results show that platelets can be harvested successfully with the Autopheresis-C system.
Materials and Methods The platelet collection method using the Autopheresis-C system has been described previ~usly.~ Figure 1 shows a diaFrom the Transfusion Medicine Center, University of New Mexico School of Medicine, and United Blood Services, Albuquerque, New Mexico; the University of Maryland Cancer Center and the Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland; the Mid Atlantic Region American Red Cross Blood Services, Norfolk, Virginia; the Hemotherapy Service, University of Arizona Health Sciences Center, Tucson, Arizona; and Fenwal Division, Baxter Healthcare Corporation, Deerfield, Illinois. Supported by grants from Fenwal Division, Baxter Healthcare Corporation. Some of the studies were conducted in the General Clinical Research Center, University of New Mexico, DRR NIHJ-MOIORR00997-15. Rcceived for publication September 24,1991; revision received January 13, 1992, and accepted January 24, 1992.
624
TRANSFUSION 1992-Vol. 32. No. 7
AUTOMATED TWO-STAGE PIATELETPHERESIS
625
is exposed to centrifugal forces as high as 300 x g (depending on the rpm). The PRP, being the less dense component, accumulates toward the center of rotation, where it exits the separation area through a series of ports that lead to the PRP outlet at the bottom of the case. Meanwhile, “packed cells” are separated from the center of the rotation where they exit the rotor through a series of packed cell ports. An integrally connected Plasmacell-C separator is provided with the kit for use in concentration of PRP. Once the desired volume of PRP is harvested, the instrument is disconnected from the donor, the separation device is discarded, and the Plasmacell-C device and tubing set are installed on the instrument. The collected PRP is concentrated to approximately 200 mL of PC; 400 to 500 mL of platelet-poor plasma is also collected as a byproduct.
In vitro studies
LJ
FIG. 1. The Plateletcell disposable set. Components on the left side of the seal are used to collect platelet-rich plasma. To the right is the platelet concentration set, installed after the donor is disconnected. PC = platelet concentrate; PPP = platelet-poor plasma; PPC = primary platelet concentrates. Reprinted with permission from Vox Sang 1990;58:100-5.
Outlet
Eleven healthy volunteers (6 men, 5 women) meeting all accepted standards for blood donors (United Blood SeMces, American Red Cross, American Association of Blood Banks, and U.S. Food and Drug Administration) participated in plateletpheresis for in vitro evaluation of collected cells. Donor age and weight ranged from 24 to 65 years and 138 to 200 pounds, respectively. Donor blood counts were performed with an automated cell counter (Coulter Electronics, Hialeah, FL) and platelet morphology score was analyzed by the methods of Kunicki5 and platelet hypotonic shock by previously described mcth0ds.l Mean platelet volume diameter and size dispersion were measured on a counter (SPlus, Coulter Electronics); donor fibrinogen, prothrombin time, and partial thromboplastin time measurements were measured by standard technique^;^ and total protein was measured by colorimetric biuret reaction (Ektachem, Eastman Kodak, Rochester, NY).The C3a component of complement was assessed by a radioimmunoassay (Amersham, Arlington Heights, IL). Protein electrophoresis as well as prothrombin time and activated partial thromboplastin time were performed on collected platelet-poor plasma. Coagulation factors V, VIII, IX, and X were measured by previously described technique^.^ Red cell and white cell counts in platelet concentrates were performed manually. Platelet aggregation testing of collected platelets used standard techniques with sinmoVL] and gle agonists (ADP at 1.8 x M [1.8 x 4.5 x 10-6M [4.5 x 10-6 mol/L], collagen at 0.17 mg/dL, and epinephrine at 0.9 x lo-‘ M [0.9 x lo-‘ moVL]) and double agonists (collagen and epinephrine at 0.15 mg/dL and 0.8 x M [0.8 x moVL], respectively, and ADP M [0.8 x and epinephrine at 1.6 and 0.8 x moV L], respectively). The extent or intensity of the aggregation reaction (total extent of primary plus secondary responses to the agonists) was expressed as a percentage of the maximum transmittance seen with the platelet-poor plasma. The slope of the reaction in degrees from the horizontal was measured along with, and the lag time from the time of addition of the agonist to the time at which aggregation started. An aggregometer (Pap 4, Biodata Corp., Hatboro, PA) was utilized. Samples were tested within 2 to 3 hours of collection.
Ro. 2. Plateletcell separator, shown diagrammatically by cutting away the surface. PRP = platelet-rich plasma. Reprinted with permission from Vox Sang 1990;58:10&5.
Anticoagulated whole blood enters the lower port of the Plateletcell device and is directed into the bottom of the rotor through a series of inlet slots. Once inside the rotor, the blood
In vivo viability studies in normal volunteers Normal volunteers meeting the same standards as volunteers for the in vitro studies participated. Paired autologous platelet recovery and survival studies were performed in five female and six male subjects aged 20 to 40. On one occasion, standard PC prepared from whole blood was studied after 5 days’ storage in a PL-732 bag; on the other occasion, platelets collected
626
with the Autopheresis-C were studied after 5 days’ storage. Radiolabeled transfusion studies were separated by a minimum of 3 weeks. One laboratory used T r radiolabeling and one used lllIndium, according to published standard methodology.s We analyzed results by both the linear and the computer-assisted gamma function methods.
In vivo platelet transjkions in patients Platelets prepared with the device were stored for up to 5 days and then provided to thrombocytopenic patients requiring transfusion. Whenever possible, we also provided pooled random-donor platelets to each patient before the test transfusion to compare the patients’ response to the test platelets (12 patients). The control transfusion was the next transfusion for patients who had received no platelet transfusion before the test transfusion. Corrected count increments (CCIs) were calculated at 0 to 2 hours (in 2 cases, at 6 hours) and 18 to 24 hours after transfusion by the following formula: CCI =
TRANSFUSION
SIMON ET AL.
Measured rise in platelet count x body surface area platelets transfused ( x loll)
Platelet counts were measured by standard techniques. We studied patients who required platelet transfusions for their clinical management; those who were clinically or serologically alloimmunized or who had splenomegaly or disseminated intravascular coagulation were excluded from the study. In all of the above studies, all donors and patients gave informed consent according to the requirements of the institutional review boards at the study locations.
Results
In vitro studies One of 11 donors had an untoward reaction, consisting of a feeling of warmth and lightheadedness with a slight drop in pulse rate without a change in blood pressure. This disappeared in 5 minutes. Donor hematologic data are shown in Table 1. There were no unanticipated changes after donation. An average of 650 mL of PRP was collected (mean, 3.85 f 0.6 x 10” platelet yield). Following concentration, the PCs had an average volume of 197 mL and contained a mean of 3.68 x 10” platelets. The platelet recovery from PRP ranged from 88 to 101 percent. Morphology scores averaged 295, as compared to 303 before concentration (maximum, 400). Hypotonic shock response did not change significantly after concentration. An average of 6.1 Table 1.
Predonation and postdonation laboratory rests in normal donors (n = 1 7 ) (mean t 1 SD)
Hematocrit (“YO) White cells x 103/pL Platelet count x 103/kL* Prothrombin time (sec) APTTt (sec) Fibrinogen (mg/dL) Total protein (gldl) C3a (ng/mL)
Predonation results
Postdonation results
46 6.0 254 12.7 37 324 6.1 258
47 6.9 180 12.8 36 295 5.6 283
f3
f 1.7
47 0.4 f4 f 63 f 0.6 2 118 f 2
f3 f 1.8
32 2 0.4 f4 f 60 f 0.4 2 131
f
* Difference between predonation and postdonation values: p
