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Vox Sang 1990;58:100-105

Platelet Collection with the Autopheresis-C@Apheresis System D. W Schoendorfer, L. H.Williamson, Ir;L. Sheckler, B. l? Fitzgerald Baxter Fenwal Division, Santa Ana, Calif., USA

Abstract. This report describes a new system for collection of platelet concentrate (PC) and cell-free plasma (PPP) from apheresis donors. The system uses two separation devices and requires only a single venipuncture. The Plateletcell@ device separates primary platelet concentrate (PPC) from anticoagulated whole blood and the Plasmacell-C@device separates the PPC into PC and PPP. Results of functional studies performed indicate that the separation process does not alter viability of either the PPC, the PC, or the PPP. Platelet function after 5 days of storage is maintained. An average yield of 3.4t0.7 x 10” platelets in 201 g of PC and 422 g of PPP were harvested in 71f13 min of donor time from donors with preprocedure hematocrits averaging 42.5+2.0% and preprocedure platelet counts averaging 265+61 x 103/pl.

Introduction Equipment and disposables that are used to perform single donor plateletpheresis have been available for almost 20 years [l]. Such specialized equipment represents degrees of complexity which restrict its use to specialized plateletpheresis centers. In addition, both the equipment and the disposables are expensive. Design criteria for development of a new plateletpheresis system include making it easy to use with little operator interaction required and, to the extent possible, retaining compatibility with existing instruments which can perform other apheresis tasks. A second area of concern in plateletpheresis is what effect separation trauma has on the platelet product. Snyder et al. [2, 31 consider separation trauma to be detrimental to the platelet product. Manual plateletpheresis requires a ‘hard’ spin of 4,900 g for 5 min to remove platelets from platelet-rich plasma. Following this separation, the platelets must be allowed to incubate quietly at room temperature for 1-2 h to allow resuspension [2]. Automated plateletpheresis procedures currently in w e can expose platelets to similar levels of trauma due to shear and centrifugal force [4, 51. Although it is impossible to draw definite

conclusions about the effect of separation trauma on the platelet product because the available data are inadequate, intuitively it would seem that separation techniques which reduce the amount of trauma involved in delivering a platelet concentrate (PC) product may provide a more hemostatically effective product. A third area of concern in plateletpheresis is related to the donor. Although there have been significant improvements in the development of ‘biologicallyclosed’ systems as well as more highly automated systems to collect platelets, very few recent improvements relate to donor comfort. A 90-min session with two phlebotomy needles in place represents the standard donor interface for continuous-flow apheresis and that interface has remained basically unchanged since the advent of single donor plateletpheresis. A system that can provide two apheresis products (PC and cell-free plasma; PPP) from a single donor will help support the goal for national self-sufficiency of plasma collection that is seen worldwide. A dual-product procedure may also help to reduce overall costs in the blood center by reducing time spent recruiting donors as well as in handling and processing the products. This report describes the performance of a new separation system - the Autopheresis-(9 apheresis system us-

Autopheresis-Ca System/Platelets

101

ing the Plateletcell@ separation device. This system addresses the issues of concern mentioned above. First, it uses a simple retrofit to the Autopheresis-C plasmapheresis instrument already in use in many centers. This instrument is considerably less expensive than existing dedicated plateletpheresis instruments and is easy to use [ 6 ] .Secondly, the Plateletcell separator was designed to provide platelets that are not exposed to detrimental cell packing during the collection process. Thirdly, the Autopheresis-C apheresis system collects a therapeutic dose of platelets in an average of 71 min from ‘average platelet donors’ using a single venipuncture. The Plateletcell device produces a primary platelet concentrate (PPC). After the donor is disconnected, the PPC is separated on the same instrument into two products (a standard volume PC and PPP) by means of an integral tube set and filtration device (the Plasmacell-C@separation device).

Materials and Methods Figure 1shows a diagram of the disposable set. The blood is anticoagulated after exiting the needle placed in the donor’s vein and then pumped into the top left side of the dual-chambered reservoir. Blood is pumped from the bottom of the left side chamber into the Plateletcell separator. Blood poor in platelets and plasma (approximately 55% hematocrit - ‘packed cells’) is pumped from the Plateletcell separator into the top of the right side of the reservoir. The anticoagulated blood flows into the left side at approximately 100 ml/min. The Plateletcell device is fed at 50 mumin allowing a reserve of blood to accumulate in the left side at a rate of 50 mumin. At the same time, the right side is filled at approximately 40 mumin with packed cells. The available reserve of blood in the left side is consumed in supplying the Plateletcell separator, while the contents of the right side are periodically returned to the donor through the same needle. This system allows the procedure to accommodate a single venipuncture for the donor (by feeding and emptying the reservoir discontinuously) while improving operating efficiency by running the separator continuously. The Plateletcell separator produces an output flow of PPC at approximately 10 ml/min. The collection continues until the desired weight of PPC is attained. A detector measures the optical characteristics of the PPC output of the Plateletcell device and modifies the rate of PPC flow and the RPM of the separator to produce maximum light absorption (i.e. maximum platelet yield) without red spectral content (i.e. RBC/WBC contamination). If the PPC redness exceeds computed thresholds, the instrument diverts that PPC into the left side of the reservoir for reprocessing. Figure 1 also shows a Plasmacell-C separator, two PL-732@plastic platelet concentrate bags and a plasma (PPP) bag with interconnecting tubing. This section of the disposable set is packaged separately from the rest of the set and hangs on a hook on the back of the machine and out of the way during the collection of PPC. Once the desired weight of PPC is harvested, the donor is disconnected. The set is sealed as marked in figure 1and that portion on

Fig. 1. Plateletcell disposable set - components on the left side of the X are used to collect PPC - consisting of the Plateletcell centrifugal separator, a dual-chambered reservoir, and a tubing harness with anticoagulant ( N C ) preconnected. To the right of the X is the PC concentration set which is installed on the Autopheresis-C instrument after the donor is disconnected from it. It consists of a Plasmacell-C membrane separator, a tubing set, and storage bags for the PC and PPP products.

the left of the seal is disposed of. The preattached Plasmacell-C device and tube set with product bags are then installed on the instrument. A command from the operator initiates a PPC concentration mode to produce a standard volume PC plus PPP. Figure 2 shows a cut-away drawing of the Plateletcell device. Centrifugal force is used to separate the blood components. The rotor contains a magnetic drive element which provides a rotational energy coupling between it and the instrument. The instrument spins the rotor to speeds up to 3,600 rpm inside the stationary case. As there are no external mechanical seals, the Plateletcell separator, like the Plasmacell-C separator, is a biologically ‘closed’ separator. 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 is exposed to centrifugal forces as high as 300g (depending on the rpm). The PPC, being the less dense component, accumulates toward the center of rotation where it exits the separation area through a series of PPC ports which \ead to the PPC outlet at the bottom of the case. Meanwhile, ’packed cells’ are separated away from the center of rotation where they exit the rotor through a series of ‘packed cell’ ports and the case through the upper ‘packed cell’ outlet.

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Schoendorfer/Williamson/Sheckler/Fitzgerald

Fig. 2. Cutaway diagram of the Plateletcell separator.

Plateletpheresis was performed on normal healthy volunteer donors (all met AABB criteria for donation) who gave informed consent. All donors denied using aspirin or other medication for 48 h prior to donation. The amount of PPC collected from each donor was determined by donor weight (600 g PPC from donors weighing 4 7 5 pounds and 700 g PPC from donors weighing 2175 pounds). Anticoagulant Acid Citrate Dextrose Formula A was used at a machine setting of 8% (1:12.5). An anticoagulated presample (pre) was drawn from an injection port in the tubing harness. Samples of the pre, PPC, PC, and PPP were subjected to laboratory analysis. One hundred grams of PC were aseptically removed at day 0 for analysis. The remaining 100 g of PC were stored in a 1,000-mlPL-732 bag on a flat bed agitator (Helmer Labs, Inc.) at 22f2"C. Samples (7 ml) were aseptically removed on day 1and day 5 for assay. Activated partial thromboplastin time (APTT), quick prothrombin time [7] (protime), factor V, factor VIII, factor IX, and factor X were determined optically (coagulation Profiler CP-8, BioiData Corp., Hatboro, Pa.) with Dade coagulation reagents. A fibrinogen assay was performed on the CP-8 using a thrombokinetic method (Thrombokinetic Fibrinogen Assay, Bio/Data Corp.) [8]. Von Willebrand factor was quantitated using an aggregometer (Platelet Aggregation Profiler PAP4, BiolData C o p ) and a macroscopic platelet aggregation test [9-111.

Platelet aggregation response was measured in a PAP4 aggregometer with single or paired agonists: adenosine diphosphate (ADP: 1.8 x 10.' M or 4.5 x 10" M ) , collagen (0.17 mg/ml), epinephrine (0.9x.' M ) . ADP (1.6~10" M ) plus epinephrine (0.8X10-5 M ) , or collagen (0.15 mg/ml) plus epinephrine (0.8 X 10" M ) [12-141. All samples for aggregations were diluted to platelet counts of 250+50 X 103/pl with plasma from a prepheresis sample. Hypotonic stress response (HSR) was determined using a modification of the method of Handin et al. [15]. The method involved combining 0.6ml of sample (250t50 X lo' platelets/$) with 0.3 ml distilled water, then recording the change in optical density for 7 min with kinetics software (DU-50 Spectrophotometer, Beckman Instruments). The control used isotonic saline in place of the distilled water.

Total protein was determined spectrophotometrically [16,171. Plasma proteins were separated electrophoretically and quantitated by means of a densitometer (Gelman Sciences, Ann Arbor, Mich.) [18, 191. IgG, IgA, IgM, and C3 were assayed using radial immunoassay D81. Hematocrits were measured by a microhematocrit technique [20]. Cell counts and sizing were determined electronically (Particle Data 80XY, Particle Data, Inc., Elmhurst, 111.) by the Electrozone method [21] or were determined manually with a hemacytomer [20]. White blood cell differentials were performed using a brilliant cresyl blue (BCB) solution. Each sample was incubated with an equal amount of BCB for 5 min, then examined in a hemacytometer. Plasma-free hemoglobin was determined by a spectrophotometric method [22]. pH at 24°C was measured with an analog pH meter (Orion Research, Inc., Boston, Mass.). Fibrinopeptide A generation was determined by a modified competitive enzyme-linked immunoassay (CELIA Diagnostica Stago, Asnieres, France) [23, 241. All data were analyzed using the corrected 'confidence interval' method for analyzing change (Texas Instruments Corp., Dallas, Tex.) All statistical comparisons are reported at ~ 5 0 . 0 5 .

Results A detailed analytical study was performed on 10 individual donors. A later qualitative study involving 96 procedures evaluated general performance characteristics. Table 1 presents the results common to both studies. There are no significant differences between the two studies. An average of 3.4 x 10" platelets with 0.5 x 10' residual white cells can be collected in an average of 71 min of donor time (needle in to needle out) from donors with an average preprocedure platelet count of 265+61 x 103/pland an average preprocedure hematocrit of 42.5 +2.0%. The procedures were well tolerated by all donors. No clinically significant physiologic changes were apparent in monitored vital signs. The donors were representative of a normal, healthy volunteer population [25].

Table 1. Procedure characteristics Parameter

Number of complete procedures PPC collection time, min PPC product weight, g Platelet yield in PC Residual WBC in PC

Analytical study 10

69f9 645f50 3.8f0.7 X 10" 0.7f0.5 X lo9

Data presented as mean k SD.

Qualitative study 96

71f 13 621f51 3.4f0.7 x 10" 0.5f0.5 X 10"

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Tables 2a and 2b highlight data from the analytical study. N o significant differences between the pre and PPP values for PT, APTT, fibrinogen, von Willebrand’s factor, or factors V, VIII :C, IX, and X are observed. Likewise, protein electrophoresis and immunoglobulin analysis show no significant difference between the pre and PPP. Total

Table 2a. Hematologic parameters, plasma product (PPP)

Parameter

Presample

PPP

Red cells, x 106/p1 White cells, x 103/pl Platelets, x 103/pl Free hemoglobin, mg/dI

4.22f0.32 6.9f1.4 253f34

Platelet collection with the Autopheresis-C apheresis system.

This report describes a new system for collection of platelet concentrate (PC) and cell-free plasma (PPP) from apheresis donors. The system uses two s...
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