Equine plasma has been recognized as a valuable resource for > 100 years. In the early 20th century, people with diphtheria infections were treated with plasma obtained from horses that had been vaccinated with diphtheria toxin, which provided passive immunity in the recipients.1 Currently, equine plasma is harvested and incorporated into culture media used by diagnostic laboratories. Equine plasma serves as the matrix in pharmacokinetic studies conducted to determine the half-life of therapeutic compounds. Highly specific antibodies used in scientific assays are isolated from equine plasma. Plasma is harvested from equine donors for transfusion into equine patients for the management of protein-losing enteropathies, nephropathies, coagulopathies, failure of passive transfer, and other medical conditions. Hyperimmune equine plasma is commercially manufactured and sold for prophylactic administration or treatment of several clinically important infectious diseases of horses.2–4 Equine plasma is the starting material in the production of hyperimmune equine fragment antigen-binding products (eg, Fab and F[ab]2), which serve as critical pharmaceuticals in the human health-care industry. These products neutralize viruses, toxins, and venoms and are often the only treatment available for life-threatening medical conditions.
Various techniques have been used over the past 100 years to harvest plasma from equine donors. Initially, investigators simply relied on the rapid erythrocyte sedimentation rate of equine whole blood to harvest the plasma.5–7 They aseptically placed large-bore stainless steel needles into jugular veins of equine donors. Tubing sets were attached to the needles, and whole blood was harvested into sterile glass collection jars containing an anticoagulant.4 Approximately 10% to 20% of the donors' blood volume was removed, which resulted in an anticoagulant-to-whole blood ratio of 1:10.4,8 After the whole blood was collected, the anticoagulated blood was refrigerated for 12 to 24 hours to allow for gravity sedimentation. After the cellular mass gravitated to the bottom of the collection jars, plasma was removed by use of a siphon or other technique. Once plasma was isolated, the remaining cellular elements were discarded. In these early investigations, plasma was harvested at intervals of approximately 30 days, which yielded 2.5 to 5 L of plasma for a typical 500-kg adult horse, without complications to the donor horses.4,7,8
In the 1970s, 2 teams of investigators each described the technique of manual plasmapheresis for harvest of horse plasma.6,8 The process involved the sequential steps of phlebotomy, fractionation, plasma extraction, resuspension of the cellular mass, and return of the resuspended mass to the respective donors.9 Whole blood was collected by use of established techniques. After gravity sedimentation for 12 to 24 hours, plasma was harvested, and the concentrated cellular elements then were resuspended in a volume of saline (0.9% NaCl) solution similar to the volume of plasma harvested. The cells and saline solution were warmed in a water bath to 37°C, then transfused back into the donors. The process was lengthy, with a considerable amount of time required for sedimentation, cellular resuspension, and transfusion. Over time, investigators began collecting whole blood in bags, which were centrifuged to substantially decrease the amount of time required for fractionation. Both teams of investigators performed manual plasmapheresis approximately every 30 days, which yielded 10 to 20 L of plasma for a typical 500-kg horse, without complication to donors.6,8
The introduction of automated in-line blood cell separators in the early 1980s revolutionized plasmapheresis procedures.9,10 These instruments attach via sterile disposable collection sets to catheters inserted in donors. Instrument pumps remove whole blood from donors and infuse anticoagulant into extracted blood at a controlled rate. The anticoagulated whole blood is then fractionated via centrifugation or filtration techniques.5,7,9 Isolated plasma is diverted to attached bags or bottles. Simultaneously, concentrated cells are returned to donors. Some of the automated instruments provide fluid replacement during collection, and others provide fluid replacement after plasma harvest is complete. Although designed for use in humans, these machines have been modified for use in automated plasmapheresis procedures of equine donors in limited settings.3,5,10,11
Automated plasmapheresis procedures offer advantages over previously used plasma harvesting methods in horses. Automated plasmapheresis procedures result in harvest of plasma in a closed system, whereas manual procedures result in harvest of plasma in an open system, which allows for possible bacterial contamination of plasma.5,7 Automated plasmapheresis procedures are a more efficient method in horses, which results in harvest of a greater volume of plasma and return of cells to donors in a shorter period.5,10 With automated plasmapheresis procedures, blood is processed through sterile disposable tubing sets and separation devices. Because the equipment is for single use only, the risk of cross-contamination to donors is eliminated, along with the need to resterilize materials.10 The automated procedure also minimizes extracorporeal deficits by simultaneous harvest of plasma and return of blood cells.9 A study5 conducted to evaluate methods for harvest of equine plasma revealed nearly complete removal of erythrocytes and leukocytes from plasma harvested via automated centrifugation. In comparison, a considerable number of erythrocytes and leukocytes remained in plasma collected via gravity sedimentation or blood bag centrifugation. Also, plasma harvested via the automated technique had greater factor VIII activity, compared with that in plasma harvested via gravity sedimentation. Investigators have evaluated automated plasmapheresis procedures for harvest of 20 mL of plasma/kg from donor horses at 30-day intervals.3,5,11 No adverse events related to the automated procedures were described in any of those reports.3,5,11
Although automated plasmapheresis represents a superior technique for the harvest of equine plasma, there is little information available on the details of the collection procedures. Companies that manufacture plasmapheresis instruments market machines specifically for use in humans, rather than in horses. Consequently, numerous logistic obstacles must be overcome before a substantial volume of equine plasma can be harvested with these instruments. Operating manuals that accompany the machines do not address automated plasmapheresis procedures in horses. Moreover, the sterile disposable collection sets and other supporting materials are designed for harvest of 500 to 800 mL of human plasma, not 20 L of equine plasma. The 3 studies3,5,11 conducted to investigate automated plasmapheresis procedures in horses include brief descriptions of the harvest techniques and indicate the instruments used for the procedures. Those reports provide valuable information regarding automated plasmapheresis procedures in horses but do not include sufficient detail to allow others to replicate the procedures.
The purpose of the study reported here was to develop and describe a closed-loop, high-speed, continuous-flow, automated plasmapheresis procedure for high-volume harvest of plasma from horses in accordance with CGMP. Current good manufacturing practices are guidelines on the manufacture of products in a high-quality system; in the United States, these guidelines are enforced by the FDA. There is continual demand for equine plasma and its constituents. Critical shortages in equine-derived antivenom products have been reported in national and international news.12–15 Availability of a reliable method for automated plasmapheresis procedures in horses may assist in meeting global demands for equine plasma and for products derived from equine plasma.
Current good manufacturing practice
Quest gel, Fort Dodge Animal Health, Fort Dodge, Iowa.
West Nile Innovator + EWT, Fort Dodge Animal Health, Fort Dodge, Iowa.
RabVac 3, Fort Dodge Animal Health, Fort Dodge, Iowa.
Fluvac Innovator EHV-4/1, Fort Dodge Animal Health, Fort Dodge, Iowa.
Pinnacle IN, Fort Dodge Animal Health, Fort Dodge, Iowa.
Life Design Prime 14, Nutrena, Minneapolis, Minn.
Autopheresis-C A-200, Baxter-Fenwal, Lake Zurich, Ill.
Champ Square CQ50L scale with CD33 indicator, Ohaus Corp, Pine Brook, NJ.
Autopheresis-C APA software for veterinary applications, version 2.0, Baxter-Fenwal, Lake Zurich, Ill.
4R-2252 Plasmacell-C set, Baxter-Fenwal, Lake Zurich, Ill.
Baxter AutoSeal tubing sealer, Baxter-Fenwal, Lake Zurich, Ill.
TSCD sterile tubing welder, Terumo Medical Corp, Somerset, NJ.
03-220-EPS1 tubing set, Charter Medical, Winston-Salem, NC.
03-220-EPS2 tubing set, Charter Medical, Winston-Salem, NC.
03-220-EPS3 tubing set, Charter Medical, Winston-Salem, NC.
EPS-20 L collection bag, Charter Medical, Winston-Salem, NC.
Dormosedan, Pfizer Animal Health, Exton, Pa.
Nolvasan surgical scrub, Fort Dodge Animal Health, Fort Dodge, Iowa.
ChloraPrep One-Step antiseptic sponge, Medi-Flex Hospital Products, Overland Park, Kan.
Hospira Inc, Lake Forest, Ill.
Becton-Dickinson, Franklin Lakes, NJ.
Supramid, S. Jackson Inc, Alexandria, Va.
Baxter-Fenwal, Lake Zurich, Ill.
Plasmalink pooling bottle, Baxter-Fenwal, Lake Zurich, Ill.
Torbugesic, Fort Dodge Animal Health, Fort Dodge, Iowa.
Plasma-Lyte A, Baxter-Fenwal, Lake Zurich, Ill.
SAS, version 9.1, SAS Institute Inc, Cary, NC.
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15 ScrippsNews. Coral snake antivenin supply is dangerously low. Available at: www.scrippsnews.com/node/32096. Accessed Jul 14, 2009.
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