A9-year-old 8.3-kg (18.3-lb) neutered male Miniature Schnauzer was referred for diagnosis and treatment of a sudden onset of lethargy, anorexia, vomiting, and pallor. On physical examination, the dog was quiet and lethargic. Mucous membranes were pale and mildly icteric, with a capillary refill time ≥ 2 seconds. Mild icterus of the skin was also noted. The dog had mild tachypnea, and cardiothoracic auscultation revealed a soft, generalized systolic murmur. Abdominal palpation elicited signs of mild discomfort, and splenomegaly was appreciated. The remainder of the physical examination findings were unremarkable.
Following IV catheter placement, blood was collected, and the dog was administered lactated Ringer solution at a rate of 90 mL/kg/d (40.9 mL/lb/d), or approximately 1.5 times the maintenance fluid rate. Quick assessment tests revealed a PCV of 17%, total solids of 6.9 mg/dL, blood glucose concentration of 153 mg/dL, and blood lactate concentration of 2.3 mmol/L. The dog was blood typed (dog erythrocyte antigen 1+a) and had macroscopic agglutination during a saline agglutination test. Results of a commercially available cage-side testb for heartworm disease and infection with Borrelia burgdorferi, Ehrlichia canis, Ehrlichia ewingii, Anaplasma phagocytophilum, and Anaplasma platys were negative. Crossmatching was not performed because of autoagglutination. Severe, regenerative anemia (Hct, 13.5%; reference range, 35.0% to 54.0%; reticulocyte count, 94.6 × 103 reticulocytes/μL) and mild thrombocytopenia (133 × 103 platelets/μL; reference range, 220 × 103 platelets/μL to 660 × 103 platelets/μL) were noted on a CBC. Anisocytosis, polychromasia, and spherocytosis were identified during cytologic examination of a blood smear. Serum biochemical abnormalities consisted of mild hyperbilirubinemia (0.4 mg/dL; reference range, ≤ 0.3 mg/dL) and hypokalemia (3.5 mmol/L; reference range, 4.0 to 4.6 mmol/L). Moderate proteinuria and microscopic pigmenturia were identified on urinalysis.
Four-view thoracic radiographs were unremarkable. Splenomegaly was apparent on 3-view abdominal radiographs, but no other radiographic abnormalities were seen. Abdominal ultrasonography revealed a heterogeneous splenic nodule (diameter, 21.8 mm), scant peritoneal effusion, and bilateral renal cortical infarcts. A presumptive diagnosis of IMHA was made on the basis of diagnostic findings.
Initial medical management consisted of corticosteroid (dexamethasone sodium phosphate, 0.15 mg/kg [0.07 mg/lb], IV, q 12 h), antiemetic (maropitant citrate, 1 mg/kg [0.45 mg/lb], IV, q 24 h), and gastroprotectant (pantoprazole, 1 mg/kg, IV, q 24 h) administration. Heart rate and rhythm were monitored by telemetry.c Twenty-four hours after initial examination, the PCV and total solids had decreased to 14% and 6.4 mg/dL, respectively. Despite worsening anemia, heart and respiratory rates, perfusion parameters, and blood pressure remained stable. No further emesis was noted. Treatment with mycophenolate mofetil (10 mg/kg [4.5 mg/lb], PO, q 12 h) was started.
On day 3, the dog became hyperpneic and developed gross pigmenturia. The PCV had decreased to 12%, and total solids was 7.0 mg/dL. The dog was transfused with 180 mL of packed RBCs. One hour after transfusion, the PCV was 18%. Because of the persistent hyperpnea, supplemental oxygen was provided by placing the patient in an oxygen cage set to a concentration of 40%. Results of repeated thoracic radiography were unchanged, and results of arterial blood gas analysis were unremarkable. Although arterial blood gas analysis did not reveal changes suggestive of pulmonary thromboembolism, treatment with dalteparin sodium (150 U/kg [68.2 U/lb], SC, q 12 h) was instituted in an effort to control possible thromboembolic disease.
Progression of the patient's clinical signs (dull mentation, jaundice, and gross pigmenturia) in combination with worsening of the macroscopic agglutination and serum hemolysis raised concerns that the dog was responding slowly or not responding to the medications chosen. Therefore, administration of a third immunosuppressant agent was commenced on day 4 (cyclosporine, 6 mg/kg [2.7 mg/lb], PO, q 24 h). In addition, the dog was transfused with 160 mL of packed RBCs because the PCV had decreased to 14% with total solids of 6.5 mg/dL. After the transfusion, the dog's PCV was 20%.
On the fifth day, the dog developed diarrhea, regurgitation, and vomiting. The patient was profoundly weak and reluctant to walk. Ondansetron (0.1 mg/kg [0.045 mg/lb], IV, q 24 h) was administered to help control nausea. Infrequent ventricular premature complexes were detected, and PCV and total solids were 15% and 4.8 mg/dL, respectively.
Because of the dog's refractory disease, a decision was made to perform a TPE procedure. Prior to TPE, the dog received a third transfusion with 120 mL of packed RBCs, after which the dog's PCV was 26%. A 7F, 16-cm-long, dual-lumen hemodialysis catheterd was then placed in the right jugular vein by means of the Seldinger technique. The pretreatment activated clotting time was measured, and the patient was anticoagulated with heparine (25 U/kg [11.4 U/lb], IV). Following administration of heparin, the activated clotting time was high, precluding the need for further heparin treatment. An exchange of 1.5 plasma volumes was performed over 180 minutes with an automated systemf and hollow-fiber TPE cartridge.g A 3% hetastarchh solution (6% hetastarch in saline [0.9% NaCl] solution) was chosen as the replacement fluid for the plasma volume exchanged during the TPE procedure. A blood sample (3 mL) was collected from the prefiltration collection port at 0, 15, and 30 minutes and every 30 minutes thereafter until termination of TPE. Posttreatment samples were collected every 12 hours for the next 2.5 days. Blood samples were allowed to clot and then centrifugedi at 500 × g for 5 minutes. Serum was harvested and transferred to sterile polypropylene cryogenic vialsj that were stored at −80°C until analyzed. The owner was informed of the relatively novel nature of the treatment and gave written consent for the TPE procedure and blood sample collection.
Twelve hours after the TPE procedure, the dog was more alert and responsive. Gross pigmenturia was markedly reduced. A coagulation panel revealed mildly prolonged prothrombin time and partial thromboplastin time, consistent with heparin treatment. Results of a CBC were suggestive of stable anemia with increased regeneration (140.6 × 103 reticulocytes/μL). Agglutination and hemolysis were markedly reduced.
On day 9, the dog was bright and alert with an improving appetite. However, abdominal palpation during physical examination of the patient elicited signs of marked abdominal discomfort. Results of abdominal ultrasonography were consistent with multifocal splenic infarcts and splenic vein thrombosis. Between a third and a half of the spleen was affected, and concerns about possible splenic necrosis prompted the decision to perform a splenectomy. Despite a stable PCV, macroscopic agglutination during a saline agglutination test persisted. Therefore, a decision was made to conduct a second TPE procedure prior to the splenectomy. Samples of patient serum were obtained and stored as previously described. No complications related to the TPE procedure were observed.
Ten days after initial examination, the dog was bright and alert with a stable PCV (26%) and total solids (4.8 mg/dL). Results of a saline agglutination test were negative, and plasma was only mildly icteric.
On day 11, the dog underwent general anesthesia for exploratory laparotomy and splenectomy. The spleen was grossly friable and necrotic in multiple areas; splenic samples were submitted for histologic examination and bacterial culture and antimicrobial susceptibility testing. The dog recovered without complications. Postoperatively, the dog was treated with methadone hydrochloride (0.2 mg/kg [0.09 mg/lb], IV q 6 h) and ampicillin-sulbactam (22 mg/kg [10 mg/lb], IV, q 8 h).
Twelve days after initial examination, the dog seemed comfortable and had a normal appetite. The previously noted ventricular premature complexes were no longer observed, likely because of removal of inflammatory splenic tissue and an increase in myocardial oxygenation. The PCV and total solids remained stable, and results of the saline agglutination test remained negative. The patient was started on clopidogrel bisulfate (2.9 mg/kg [1.3 mg/lb], PO, q 24 h).
On day 13, there was no evidence of hemolysis or agglutination. A CBC was performed, but regeneration was not observed (61.8 × 103 reticulocytes/μL). The patient's WBC count had markedly increased (68.5 × 103 WBCs/μL; reference range, 8.0 × 103 WBCs/μL to 14.5 × 103 WBCs/μL), and toxic neutrophils were observed during cytologic examination of a blood smear. Although the cause was not entirely certain, preliminary results of histologic examination of splenic samples were suggestive of an inflammatory process. A blood sample was submitted for bacterial culture, and treatment with enrofloxacin (15 mg/kg [6.8 mg/lb], PO, q 24 h) was started.
On day 14, the dog was discharged from the hospital. At the time of discharge, the PCV and total solids were 28% and 5.4 mg/dL, respectively. In addition, administration of dalteparin sodium was discontinued, and the dog was being treated with prednisone (1.2 mg/kg [0.54 mg/lb], PO, q 12 h), amoxicillin-clavulanic acid (13.75 mg/kg [6.25 mg/lb], PO, q 12 h), enrofloxacin (15 mg/kg, PO, q 24 h), clopidogrel bisulfate (2.9 mg/kg, PO, q 24 h), cyclosporine (6 mg/kg, PO, q 24 h), and mycophenolate mofetil (10 mg/kg, PO, q 12 h).
Two days after discharge, the dog was reexamined, and results of a follow-up CBC were suggestive of regenerative anemia (153.0 × 103 reticulocytes/μL) and progressive leukocytosis (83.2 × 103 WBCs/μL). Results of histologic examination of splenic samples were most consistent with suppurative splenitis, and bacterial culture of a blood sample did not yield any aerobic or anaerobic growth. A decision was made to discontinue one of the immunosuppressant medications because of concern for sepsis related to suppurative splenitis, and a decision was made to discontinue cyclosporine administration because it has been associated with an increased risk of fungal dermatopathies among animals in subtropic regions.
Three months after discharge, prednisone administration was discontinued. The dog continued to receive mycophenolate mofetil as the sole immunosuppressant medication.
Immunoglobulin concentrations in the previously stored serum samples were measured by use of a commercially available radial immunodiffusion system in accordance with the manufacturer's directions.k Briefly, the radial immunodiffusion plates consisted of a homogeneous agarose gel containing either anti-canine IgG or anti-canine IgM with preformed circular wells for deposition of patient serum. Standardized quantities of the patient's serum were pipetted into the preformed circular wells, and the diameter of the precipitin ring caused by immunoglobulins reacting with the embedded antibodies was measured to the nearest 0.1 mm with a calibrated loupe 3 times and averaged. A standard curve to calculate immunoglobulin concentrations was determined by means of reference sera,k with the logarithm of reference serum concentration (x-axis) plotted against ring diameter (y-axis). A fitted line was determined by means of least squares regression analysis. All plates were run in triplicate, and results were averaged.
During the first 3-hour TPE procedure, serum IgG concentration decreased by 68% (Figure 1), and 24 hours after the procedure, serum IgG concentration was still 24% less than the pretreatment (time 0) concentration. By 48 hours after the procedure, however, serum IgG concentration had nearly rebounded to the pretreatment concentration. During the second TPE procedure, serum IgG concentration decreased by 47% and returned to the pretreatment concentration within 12 hours.
Plasma IgG concentrations during and after TPE procedures performed on days 5 (diamonds) and 9 (squares) in a 9-year-old Miniature Schnauzer with refractory IMHA. The error bars represent the SEM for the repeated immunoglobulin concentration measurements.
Citation: Journal of the American Veterinary Medical Association 252, 9; 10.2460/javma.252.9.1108
During the first TPE procedure, serum IgM concentration decreased by 64%, and it was 31% less than the pretreatment concentration 24 hours later (Figure 2). Serum IgM concentration decreased from that point and, 60 hours after the procedure, was only 56% of the pretreatment concentration. During the second TPE procedure, IgM concentration decreased by 64% and was still decreased by 26% at 12 hours.
Plasma IgM concentrations during and after the first (diamonds) and second (squares) TPE procedures for the dog in Figure 1. The error bars represent the SEM for the repeated immunoglobulin concentration measurements.
Citation: Journal of the American Veterinary Medical Association 252, 9; 10.2460/javma.252.9.1108
Discussion
Therapeutic plasma exchange is an extracorporeal blood purification technique whereby unwanted plasma substances are separated from the cellular constituents of blood.1 Autoantibodies, immune complexes, and toxins are removed and subsequently replaced by a colloid solution, such as plasma, albumin, or synthetic colloids.1 Use of fresh frozen plasma has been associated with high morbidity and mortality rates in human patients and therefore is discouraged.2 In people, 4% to 5% human albumin diluted in saline solution is the most commonly used replacement solution.3 Unfortunately, canine albumin is unavailable, and the use of human albumin in dogs has been associated with adverse clinical reactions, including anaphylactoid reactions, serum biochemical abnormalities, severe edema, and production of anti-albumin antibodies.4 Therefore, we chose to use a dilute (3%) hetastarch solution. Although use of synthetic colloid solution has raised concerns about kidney injury in people, there were at the time of the report presented here no prospective studies demonstrating that this concern applied to dogs.5
Therapeutic plasma exchange can be performed by either filtration or centrifugation techniques; however, separation through filtration is more common in veterinary medicine.6 With filtration TPE, the patient's blood enters a dialyzer, where it is divided into thousands of straw-like semipermeable membranes.7 The blood is exposed to a positive transmembrane pressure that causes the plasma to be forced out so that it can be discarded. When plasma is discarded, proteins (albumin and globulin) and protein-bound substances are efficiently removed from the bloodstream. Meanwhile, the cellular components of blood are mixed with a colloid solution and returned to the patient.
Removal of any substance (eg, antibodies) from the blood by TPE is limited. As the patient's plasma is being removed and exchanged for a replacement solution, the substance of interest remaining in the patient is being diluted. Although an exchange of 1.5 plasma volumes may result in a 60% to 70% reduction in antibody concentrations, additional exchanges become increasingly less efficient. Because of this, current recommendations are that TPE be limited to 1.5 plasma volume exchanges.3
Therapeutic plasma exchange has been used in human medicine for removal of certain toxins as well as for treatment of specific immune-mediated diseases.1,8–13 The American Society for Apheresis has established guidelines for the use of TPE in human clinical practice. Diseases are sorted into 4 categories and 2 grades on the basis of the role TPE has in treating each disease and the quality of evidence supporting its use.3 Under this grading scheme, TPE is accepted as a second-line treatment for IMHA in people either when traditional treatments fail or in conjunction with traditional treatments.3
In people, IMHA is known to behave in 1 of 2 ways. In warm antibody disease, IgG is the predominant antibody that targets and destroys RBCs through complement-mediated lysis or via the extravascular macrophage-phagocytotic system.8 Cold antibody disease tends to be associated with highly specific medical procedures (eg, certain cardiac procedures) during which an induced decrease in core body temperature triggers IgM autoantibody production and subsequent targeting of RBCs.8 Unlike IgG, which is mainly found within the extravascular space, approximately 78% of IgM is found in the intravascular space.14 Because of this, diseases involving IgM may be more responsive to TPE,1,8,14,15 and studies6,8,15 have shown a greater decrease in IgM than in IgG concentration when performing TPE, as would be expected on the basis of antibody distribution. Nevertheless, TPE seems beneficial in patients with either warm antibody or cold antibody IMHA.8
Although TPE is commonly thought to work by decreasing the severity of immune-mediated destruction of cells through removal of circulating autoantibodies and immune complexes, other mechanisms of action have been proposed. Studies1,14–16 have demonstrated that following TPE, antibody concentrations return to baseline concentrations (rebound) and sometimes even surpass baseline concentrations (overshoot) despite clinical improvement in the patient. This suggests that TPE may have immunomodulatory effects at the cellular level, with initial removal of antibodies resulting in a proliferation of immune cells, thereby making them more susceptible to cytotoxic pharmaceuticals.17 Additionally, TPE has been shown to directly modulate immune function, resulting in a shift in T-helper cell balance and promoting changes in expression of interleukin, interferon, and other cytokines.1,18 The antibody rebound and immunomodulatory effects provide support for pulsatile TPE treatment. With this method, patients receive several TPE treatments spaced 24 to 48 hours apart.1 Although antibodies return afterwards, they appear less directed at autoantigens.
The quality and quantity of the veterinary literature supporting the use of TPE for treatment of patients with immune-mediated diseases are currently deficient, and we are aware of only 4 case reports6,17,19,20 describing the use of TPE for the treatment of immune-mediated diseases in dogs, including dogs with IMHA, myasthenia gravis, and systemic lupus erythematosus. In only one of these reports,6 which involved a single TPE treatment for a dog with IMHA, were immunoglobulin concentrations measured. However, the method for measurement of immunoglobulin concentrations was not explained, and measurement of immunoglobulin concentrations stopped at the end of the single TPE treatment. This did not allow for documentation of clinical disease improvement despite a return of antibodies after treatment, as appears to occur in people and other animals.
For the dog described in the present report, serum IgG and IgM concentrations were measured during and after each TPE procedure. Both concentrations decreased during the procedure, but rebounded to pretreatment concentrations within 36 to 48 hours. However, despite the rebound in antibody concentrations, the patient maintained a stable PCV and improved clinically following TPE. To the authors' knowledge, this is the first clinical case providing evidence that TPE may have the same immunomodulatory effects in dogs as have been proposed to occur in people and laboratory animals. Further, our findings suggested that TPE may be a useful alternative in dogs with refractory IMHA when traditional treatments have failed.
ABBREVIATIONS
| IMHA | Immune-mediated hemolytic anemia |
| TPE | Therapeutic plasma exchange |
Footnotes
Quick Test DEA 1, Alvedia Veterinary Diagnostics, Limonest, France.
SNAP 4Dx Plus, Idexx Laboratories Inc, Westbrook, Me.
VST Transmitter, DRE Medical, Louisville, Ky.
Mila International, Florence, Ky.
Heparin sodium injection USP, Sagent Pharmaceuticals, Schaumburg, Ill.
Prismaflex, Gambro Industries, Meyzieu, France.
Gambro TPE 200 SET, Gambro Industries, Meyzieu, France.
Hospira Inc, Lake Forest, Ill.
LWS M24 Combo Microhematocrit Combo Centrifuge, LW Scientific Inc, Atlanta, Ga.
T309-2A Cryovial, 2 mL, Simport, Beloeil, QC, Canada.
Kent Laboratories Inc, Bellingham, Wash.
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