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Apheresis in Veterinary Medicine: Therapy in Search of a Disease?

INTERNATIONAL VETERINARY EMERGENCY
AND

CRITICAL CARE SYMPOSIUM 2009

Reid Groman, DVM, DACVIM, DACVECC University of Pennsylvania, Philadelphia, PA, USA
19136116 The word apheresis is Greek for "to take away". In all apheresis procedures, blood is removed from a patient or donor, and blood separator technology is used "to take away" a specific cell type or plasma constituent. Nomenclature varies but generally describes the specific blood element that is removed, e.g., plasmapheresis, leukocytapheresis, erythrocytapheresis, and plateletpheresis. In human therapeutic apheresis (TA), specific blood components are removed and replaced (e.g., red cell exchange for sickle cell anemia) or removed and discarded (leukoreduction for hyperleukocytosis, plateletpheresis for thrombocythemia). For donor apheresis, blood is taken from a healthy individual, the needed component is collected, and the remaining components are returned to the donor. Donor apheresis can be used for the collection of platelets, plasma, granulocytes, and red cells, as well as for harvesting peripheral blood stem cells (PBSCs). Donor apheresis is the major procedure performed in human medicine, and is performed at some veterinary blood banking facilities.

Blood components are separated by apheresis equipment using centrifugation to separate by weight (specific gravity) or filtration to separate by size. The centrifugal method is used primarily in North America, whereas membrane separation is mostly used in Europe and Japan. In centrifugal separation, whole blood is separated into various components based on specific gravity of the components. Blood is fed continuously into a rapidly rotating bowl (in essence, a large centrifuge) where components are separated, the desired layer or layers removed, and the remainder returned to the patient with replacement fluid. With filtration technology, removed blood is pumped through highly permeable membranes. Plasma readily passes through the membrane pores (0.2-0.5 m; MW cutoff ~ 2000 kDa) while the cells are simultaneously retained and returned to the patient. Membrane filtration can be done using hemodialysis equipment, including the Gambro PrismaTM and PrismaFlexTM. A further technical refinement involves specific removal of a plasma component. After initial separation of plasma from cells, the separated plasma can be processed by a second membrane, also called a plasma fractionator, to remove specific macromolecules or immune complexes, a procedure known as double filtration or cascade filtration. The pore diameter of the second membrane is as small as 0.03 m, and the plasma components that cannot pass through this filter are discarded, while the cleared plasma is recombined with cells and returned to the patient. This technique is not widely used in North America. Both centrifugation and membrane apheresis require anticoagulation to prevent activation of the coagulation system within the extracorporeal circuit. The most frequently used anticoagulant for centrifugation procedures is acid-citrate-dextrose (ACD) solution, given as a continuous infusion with infusion rate adjusted according to the blood flow rate. Citrate binds calcium, thus removing it from the coagulation cascade. In many human apheresis procedures (stem cell collections, platelet donation) calcium does not need to be replaced. In companion animal TA, most procedures using citrate for anticoagulation are safely performed when calcium is given to the patient independent of the circuit. Initial administration of 50-100 mg/kg/hr calcium gluconate is recommended, with adjustments based on periodic ionized calcium analysis and/or patient symptoms. This recommendation from the human literature corroborates our experience with citrate in dogs, but there can be wide variability depending on the citrate infusion rate and the blood volume of the patient. Infrequently, clinical signs of hypocalcemia such as agitation and lip licking are observed in dogs and resolve promptly with calcium repletion. Standard unfractionated heparin is most frequently used for membrane apheresis. The required dose is often higher than prescribed for hemodialysis patients because a significant amount of infused heparin is removed along with the plasma. An initial loading dose of heparin (40 U/kg IV) is followed by a continuous infusion (20-40 U/kg/hr IV) and adjusted to maintain adequate anticoagulation in the circuit. In veterinary medicine, ACT is measured every 30 minutes. Many human apheresis centers successfully utilize algorithms to guide heparin dosing without need for ACT monitoring. "Custom" combinations of citrate and heparin are occasionally used in human apheresis procedures. A central venous catheter is used for veterinary apheresis procedures at the University of Pennsylvania although some human procedures require only antecubital access. A standard hemodialysis catheter may be used, or a single lumen central catheter may be used for blood removal, while a peripheral vein can be used for return through a standard 18 gauge catheter. Blood flows are typically slower than those for hemodialysis and as such smaller diameter catheters may be inserted for both therapeutic and donor procedures. The smallest extracorporeal circuits are 165-185 mls, making it challenging to maintain hemodynamic stability in small animals. When blood is used for
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"priming" the extracorporeal circuit, human infants tolerate apheresis without apparent consequence. Blood priming should be considered in anemic patients, and any time the extracorporeal blood volume exceeds 15-20% of the patient's total blood volume. In human medicine, clinical applications of therapeutic apheresis (TA) were initially based on anecdotal and uncontrolled studies. In the last two decades, more rigorous examination has reaffirmed its place in the management of a number of disorders, and the American Society for Apheresis has published evidence based guidelines. In small animal practice, it is unknown if TA is indicated for any disease or syndrome at this time, with the literature limited to single case reports or case series that suggest efficacy. In order for TA to be rationally applied, three conditions should ideally be met: 1) the disease or symptom is caused by a blood constituent that can be depleted, 2) TA is shown to meaningfully deplete the offending constituent, and 3) documented depletion is accompanied by clinically significant benefit. It is often not possible to apply all three of these conditions to human diseases at present, let alone a veterinary disease, and the small body of existing literature (e.g., preclinical human studies using dogs as models, sporadic case reports in dogs) can only be used as a guideline for identifying patients that may be rationally treated with TA, and as a springboard for designing further studies of donor apheresis for transplantation and oncology. By definition, plasmapheresis is the separation of plasma from blood cells. Removal of large volumes of plasma requires replacement solutions and the term therapeutic plasma exchange (TPE) is more appropriate. The terms are often used interchangeably in the literature. TPE, non specific plasma removal through cell separation, depletes the patient of all plasma constituents including clotting factors and immunoglobulins, and colloid solutions are used for replacement in order to maintain oncotic pressure. The most common replacement solution used in human TPE is 5% albumin. Less commonly, fresh frozen plasma is used for this purpose. There are several mechanisms by which plasmapheresis exerts its beneficial effects, most notably by the rapid depletion of specific diseaseassociated factors including pathologic autoantibodies, immune complexes, and paraproteins. TPE also allows the infusion of "normal" plasma, which may replace a deficient plasma component, coagulation factors and nonpathogenic immunoglobulins. In practice, documented utility of TPE to bring about lasting depletion has been limited to large molecules, e.g., antibodies. "Cleansing" the blood of smaller, shorter lived molecules, including various mediators of inflammation is not substantiated by meaningful depletion or borne out by clinical studies. The rationale for using TPE in clinical toxicology should be confirmed in each type of intoxication by evidence of effective clearance, as well as by high plasma protein binding (>80%) and a low volume of distribution of the toxic substance (<0.21/kg BW). TPE is used to treat intoxication with phalloid mushrooms. Although the most potent amatoxins ( and -amatine) appear to be dialyzable, there is no benefit if hemodialysis is initiated after the first few hours post-ingestion. TPE has the advantage of eliminating these highly protein bound toxins and their endogenous metabolites, and reduces mortality in humans (<20%) compared to conventional therapy (30-50%), even when severe clinical signs are present. Case reports and small case series show TPE to be effective in life threatening intoxications with tricyclics (amitriptyline), L-thyroxine, verapimil, diltiazem, and theophylline. Adjunctive TPE may be used to manage patients with life-threatening dermatoses. Pemphigus foliaceous is a potentially fatal disease characterized by autoantibodies against cell surface adhesion proteins. Erythema multiforme (EM) is a skin disease of unknown etiology, mediated by deposition of immune complexes in the microvasculature of the skin and oral mucous membranes that usually follows an antecedent infection or drug exposure. In its most severe form, EM major, use of TPE is described for patients with this life-threatening disorder. Similarly, some evidence supports TPE for human pemphigus patients who do not respond to conventional immunosuppressive regimens. Both EM and pemphigus are well described in the veterinary literature. There is little information about use of TPE in the management of dermatoses in dogs and cats at this time. However, given the immunologic basis of the diseases, consideration may be given to severely affected dogs when the disease cannot be controlled with medical management. Generally considered a salvage procedure in critically ill septic patients, recent animal studies and clinical trials suggest that TPE may have an adjuvant therapeutic role in the management of severe sepsis. The theoretic rationale is that TPE will remove inflammatory mediators, endotoxin, deleterious antibodies and immune complexes. The reason for possible improvement is unclear, and multicenter trials evaluating valid clinical outcomes (mortality or organ failure), rather than surrogate markers such as mediator clearance or transient improvement in physiologic variables, are required to define the precise role of extracorporeal therapies in the management of sepsis. TPE also allows the infusion of normal plasma, which may replace a deficient plasma component, and may be among the mechanisms of action in specific disease syndromes, including severe sepsis. Dysproteinemias consist of a broad range of serious diseases in with the common thread of excessive production of an abnormal, or paraprotein, generally immunoglobulin. In humans, TPE in conjunction with other therapies has
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been used widely to treat dysproteinemias and their resulting clinical syndromes. Syndromes include hypervolemia, renal failure and neuropathies; symptoms are ascribed to poor delivery of oxygen under conditions of sluggish capillary flow. Hyperviscosity syndromes due to abnormal plasma components are seen infrequently in veterinary medicine, but case reports describe the use of plasmapheresis in dogs and cats with multiple myeloma, and in one dog with E canis associated hyperglobulinemia Hyperviscosity may be due to excessive circulating blood cells, e.g., polycythemia, or more commonly hyperleukocytosis associated with acute leukemia. In humans, cytoreductive procedures (leukocytapheresis) may be performed on an emergent basis for these syndromes. Recently we treated a cat with severe refractory hyperviscosity/mixed cryoglobulinemia associated with plasma cell pododermatitis with manual plasma exchange. The cat suffered from central and peripheral neurologic signs and renal failure. Preprocedure, the cat's blood was too viscous to be collected through a 22 gauge needle. Following each exchange (60 mls every 24 hours), neurologic status improved significantly, including activity level and appetite. Renal function did not improve, however, and the cat was euthanized. Immune mediated hemolytic anemia (IMHA) is often caused by circulating IgG or IgM, providing a plausible rationale for TPE. Most cases are due to IgG antibodies that promote red cell destruction by Fc receptor bearing phagocytes in reticuloendothelial organs. In human medicine, TPE has been used sporadically in cases of autoimmune hemolytic anemia for many years. Most published results have appeared as case reports in which reduction in antibody titers has been achieved but clinical response has been inconsistent. Concomitant therapy with immunosuppressives complicates interpretation of favorable outcomes. Some authors feel that TPE helps to stabilize patients with fulminant hemolysis or improve responsiveness to transfusions, while others believe that TPE is ineffective for this purpose. While the rationale seems sound, human and veterinary data are limited to case reports. It is possible that the veterinary experience would be quite different, and at present TPE for adjunctive management of severe canine IMHA should not be categorically written off. It may be possible to document clinical improvement, and to correlate this with pre- and post-procedure direct and indirect Coombs' titers in patients failing to respond to immunosuppressive drugs. Immune mediated thrombocytopenia (IMT) often responds favorably and durably to immunosuppressive drugs, but TPE may be considered in refractory cases. It is thought that pre and post-pheresis platelet associated IgG levels can be measured, and the results correlated with clinical response. TPE is indicated for several neurologic and glomerular diseases, including Guillain-Barre, demyelinating polyneuropathies, myasthenia gravis (MG), and several rapidly progressive glomerulonephritides. Fulminant MG is a Class I indication for TPE in man, and several case reports describe the successful implementation of TPE for dogs with this condition. The etiology of acute inflammatory polyradiculoneuropathy (AIP, Coonhound Paralysis) is not firmly established but immune mediated mechanisms are strongly suspected. The pathogenic mechanisms of this disease are sufficiently similar to human Guillain Barre syndrome that TPE might be effective in dogs not responding to conventional therapy. Few canine glomerular diseases are treated with immunosuppressive therapies. There is now compelling evidence to support the immunopathogenesis of Lyme-associated glomerulonephritis, and while the indications for hemodialysis are not clear in this setting, TPE may be used in an effort to eliminate circulating antibodies and immune complexes, in addition to current standards of care (e.g., ACE inhibition, antibiotics). Interestingly, ACE inhibitors are often discontinued when TPE is performed in humans, in order to avoid severe bradykinin reactions. Despite heightened awareness and several recent publications, relatively little is known about this syndrome. There are no studies specifically examining the role TPE in canine glomerular diseases, although the addition of immunosuppressive drugs to conventional medical management is currently being studied in a prospective manner. Collection of peripheral blood stem cells (PBSC) as a source of hematopoietic stem cells is steadily increasing in veterinary oncology. The principle of hematopoietic cell transplantation is to destroy the malignant cells with high dose chemotherapy and/or irradiation and thereafter restore the bone marrow with stem cells. After six days of GCSF therapy to drive CD34(+) stem cells from bone marrow into the peripheral blood, canine PBSCs are harvested using a continuous centrifugation separation device (Baxter CS 3000TM or Cobe SpectraTM) and given to the patient immediately after total body irradiation. Currently, autologous bone marrow transplants are performed in dogs weighing > 18 kgs with high grade lymphomas. DLA matched siblings or parents may be used for allogeneic transplants. Ultrapheresis is a variant of membrane filtration that has been investigated in veterinary cancer patients. Ultrapheresis removes immunosuppressive factors in circulation, e.g., soluble TNF receptor, and thereby and in theory reverses immunologic tolerance to the cancer neoantigen. Although to the author's knowledge ultrapheresis is not presently used in veterinary patients, preliminary findings in tumor bearing dogs were encouraging. Current veterinary transplant immunology studies employ leukoapheresis to remove specific regulatory T cells. These cells, once identified by flow sorting, can be expanded ex vivo, manipulated, and reintroduced to the transplant recipient.

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Platelet transfusions are indicated for the management of life threatening hemorrhage due to thrombocytopenia or thrombopathia. Fresh whole blood, platelet rich plasma, and platelet concentrates are most commonly used for platelet transfusion in most veterinary settings. A major advantage of apheresis platelets is that enough platelets can be collected from a single donor to constitute a transfusion "dose". Plateletpheresis may be performed with several automated instruments, and three different automated separators have been used for collecting platelets from healthy donor dogs. All machines differ in design and configuration, but all use centrifugal force to separate blood components, and all provide an acceptable platelet yield. We have documented that plateletpheresis can be safely and reliably performed in healthy dogs weighing > 20 kgs using the Cobe Spectra (Caridian BCT)TM. Platelet concentration was 1.4 x 106 platelets/l. The total platelet yield, in a total collect volume of 240 mls, is typically 3.3 x 1011. We administered fresh (within 30 minutes of collection) platelet concentrate to a dog with amegakaryocytic IMT and severe life-threatening bleeding five days after aggressive immunosuppressive therapy and multiple transfusions. Subsequently, all evidence of bleeding ceased and no further transfusions were needed. It is assumed that transfused platelets will have a reduced lifespan, especially in the setting of IMT. Donor procedures can be performed on an outpatient basis. Therapeutic apheresis procedures may be performed cageside or in a dedicated treatment area. The major complications are usually related to the relatively large extracorporeal blood volume, including temperature changes, hypocalcemia, hypotension, and rarely other electrolyte disturbances. These are all identifiable and more important preventable in order to make this a safe modality for small dogs and cats. The indications for performing therapeutic apheresis in companion animals are not defined, the present literature consisting of case reports and case series. While there are a small number of candidate diseases, and a limited number of equipped facilities, it is anticipated that prospective controlled studies will support apheresis for patients with life-threatening presentations of neoplastic and immunologic diseases. References
1. Bartges JW. Therapeutic plasmapheresis. Sem Vet Med Surg 1997;12:170-177. 2. Brecher ME. Plasma exchange: Why we do what we do. J Clin Apheresis 2002;17:207-211 3. Callan MB, Appleman EH, Shofer FS, et al. Clinical and clinicopathologic effects of plateletpheresis on healthy donor dogs. Transfusion 2008;48:2214-2221 4. Hale AS. Clinical applications of automated apheresis. 2007 Proceedings of the American College of Veterinary Internal Medicine 5. Lucas RL, Lentz LVT, Hale AS. Collection and preparation of blood products. Clin Tech Small Anim Prac 2004;19:5562 6. Salma A, Kieswetter H, Kalus U. Massive platelet transfusion is a rapidly effective emergency treatment in patients with refractory autoimmune thrombocytopenia. Thromb Haemost 2008;100:762-765 7. Szczepiorkowski ZM, Bandarenko N, Kim HC. Guidelines on the use of therapeutic apheresis in clinical practice-evidence based approach from the Apheresis Applications Committee of the ASA. J Clin Apheresis 2007;22:106-175

SPEAKER INFORMATION

(click the speaker's name to view other papers and abstracts submitted by this speaker) Reid Groman, DVM, DACVIM, DACVECC University of Pennsylvania Philadelphia, PA

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