Presentation

A 5 yr old spayed female border collie mix weighing 31.8 kg presented to her primary care veterinarian for anorexia and lethargy of 1 day duration. On physical examination, the dog was tachycardic with a heart rate of 128 bpm and febrile with a temperature of 40.3°C. Splenomegaly was noted on abdominal palpation. The remainder of the physical examination was unremarkable.

Diagnostic Investigation

A complete blood count revealed a mild decrease in hematocrit (33% [reference interval (RI) 36–60%]), mild decrease in hemoglobin (11.5 g/dL [RI 12.1–20.3 g/dL]), moderate macrocytosis (87 fL [RI 58–79]), mild hyperchromasia (30.3 pg [RI 19–28 pg]), and a moderate rubricytosis (4 nucleated red blood cells per 100 white blood cells [RI 0–1/100 white blood cells]). A white blood cell differential revealed a mild lymphopenia (657 lymphocytes/µL [RI 690–4500/µL]). Microagglutination (no saline dilution was performed to rule out rouleaux formation), slight spherocytosis, and moderate polychromasia were observed on blood smear examination by a boarded clinical pathologist. The corrected reticulocyte count was 2% (considered an appropriate response if above 1% in the dog) with an absolute reticulocyte count of 110,200/µL (RI <60,000/µL) indicating moderate red blood cell regeneration. On serum biochemistry there was mild hyperbilirubinemia (0.7 mg/dL [RI 0.1–0.3 mg/dL]) and mild hypokalemia (3.4 mEq/L [RI 3.6–5.5 mEq/L]). A total T4 was within normal limits. On urinalysis, the urine was moderately alkaline (pH 7.5 [RI 5.5–7.0]) with mild proteinuria (1+) and a specific gravity of 1.037. An occult heartworm antigen test was negative. Abdominal radiographs were performed, revealing splenomegaly, which was further confirmed via ultrasound.

The following day, the results of a polymerase chain reaction (PCR) canine tick-borne panel profile through Antech Diagnostics were available and were negative for all organisms tested including Anaplasma phagocytophilum, A platys, Babesia canis, Babesia spp. (noncanis), Bartonella henselae, B vinsonii, E canis, Erlichia spp., Mycoplasma haemocanis/haematoparvum, Neorickettsia risticii, and Rickettsia rickettsii. Serum samples were subsequently submitted to the North Carolina State University vector borne diagnostic laboratory for immunofluorescent assay for B canis and B gibsoni and PCR for Babesia and Bartonella spp., to Galaxy Diagnostics for PCR and culture for Bartonella spp., and to the Colorado State University veterinary diagnostic laboratory for PCR for M haemocanis and M haematoparvum. Results were all reported as negative.

Therapy

Initial therapy, commenced by the primary care veterinarian while awaiting further diagnostics, focused on treating potential tick-borne illness and possible immune-mediated hemolytic anemia (IMHA). The patient was prescribed oral doxycycline (5 mg/kg per os [PO] q 12 hr, 10 day course) and prednisone (1 mg/kg PO q 12 hr).

Given the presence of spherocytosis and microagglutination accompanying a regenerative and apparently hemolytic anemia, the negative test results for infectious disease, and the lack of evidence of other underlying disorders, a provisional diagnosis of primary IMHA was established. Based the recently published American College of Veterinary Internal Medicine consensus statement on the diagnosis of IMHA, the dog would have been classified with a diagnostic category of “Supportive of IMHA,” with one sign of immune-mediated destruction (spherocytes), and one sign of hemolysis (hyperbilirubinemia).⁴ One day after presentation, oral azathioprine (1.6 mg/kg PO q 24 hr) was prescribed, along with aspirin (0.6 mg/kg PO q 24 hr) and famotidine (1 mg/kg PO q 24 hr) in an attempt to reduce the likelihood of pulmonary thromboembolism and gastric ulceration, respectively. Over the following 2 days, the hematocrit continued to decrease to 22%; therefore, the azathioprine dose was increased to 1.6 mg/kg PO q 24 hr for 5 days/wk and 1.6 mg/kg PO q 12 hr for 2 days/wk (Wednesday and Saturday), for an average daily dose of 2 mg/kg. Minimal improvement in hematocrit was observed with immunosuppressive therapy over the next 2 wk; therefore, compounded cyclosporine^a (3.1 mg/kg PO q 12 hr) was added concurrently to the prednisone and azathioprine in an effort to increase the degree of immunosuppression.

Over the next 4 days, the patient began acting more lethargic and having soft stools. Azathioprine was discontinued, prednisone dose rate was decreased (from 1 mg/kg to 0.7 mg/kg PO q 12 hr), and cyclosporine dose rate was increased to 3.1 mg/kg PO q 8 hr. S-adenosylmethionine was commenced as a potential hepatoprotectant owing to elevations in liver enzymes (aspartate aminotransferase 76 U/L [RI 15–66 U/L], alanine aminotransferase 280 U/L [RI 12–118 U/L], alkaline phosphatase 1938 U/L [RI 5–131 U/L], and gamma-glutamyl transferase 232 U/L [RI 1–12 U/L]) detected on repeat serum chemistry. Two days after starting the new regimen, hematocrit had further decreased to 21%, and other measures of disease activity (including spherocytosis and hyperbilirubinemia) had persisted; therefore, prednisone dose was increased back to 1 mg/kg PO q 12 hr and an additional immunosuppressive agent, mycophenolate mofetil (10 mg/kg PO q 12 hr), was added. Two days later, cyclosporine dose was adjusted to 4.7 mg/kg PO q 12 hr.

Five days later, the patient developed an open wound with a draining clear discharge on her right lateral hip, which was hypothesized to have been due to infection secondary to significant suppression of the immune system. Mycophenolate mofetil was discontinued, and amoxicillin/clavulanic acid (27.5 mg/kg PO q 12 hr) and enrofloxacin (2.2 mg/kg PO q 12 hr) were commenced. The lesion did not improve over the following 48 hr; therefore, prednisone and cyclosporine dosage rates were decreased to 1 mg/kg PO q 24 hr and 3.3 mg/kg PO q 12 hr, respectively, and melatonin (0.1 mg/kg PO q 12 hr) was commenced as a potential immunomodulating agent.⁵ Although the draining lesion healed over the following week, the patient developed cystic and exudative lesions on the dorsorostral muzzle, tail, and perianal region, which then improved over the next 3 days with continued antibiotic therapy.

Outcome

Given the ongoing challenges associated with maintaining a level of immunosuppression that controlled IMHA without resulting in suspected secondary infection, the primary care veterinarian sought to fine-tune the cyclosporine doses by sending a blood sample to the Mississippi State University (MSU) Pharmacodynamic Laboratory for measurement of activated whole blood expression of interleukin-2 (IL-2) messenger RNA (mRNA) using a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay.^2,3,6–9 On a blood sample collected approximately 2 hr after administration of the morning dose of oral cyclosporine, IL-2 mRNA expression was found to be markedly suppressed (0% of control samples). The MSU Pharmacodynamic Laboratory defines “marked” suppression of IL-2 expression as less than 5% of cytokine expression of normal control samples. This was an unexpected result given the relatively low dose of cyclosporine the patient was on at the time of submission (3.3 mg/kg q 12 hr). Typically, marked suppression of cytokine levels is most often observed by the MSU Pharmacodynamic Laboratory when patients are on significantly higher cyclosporine dosages (unpublished data): a starting dose of 5 mg/kg orally q 12 hr is typically recommended for systemic immunosuppression in dogs, with the expectation that, in most dogs, this will cause suppression in the moderate range or higher when dogs are dosed with the standard modified (microemulsified) preparation of cyclosporine. The MSU Pharmacodynamic Laboratory defines “moderate” suppression of IL-2 expression as between 25 and 50% of cytokine expression of normal control samples, and “moderate to high” as between 5 and 25%.

Given the high level of IL-2 suppression in response to a relatively low cyclosporine dosage, 3.3 mg/kg q 12 hr, a recommendation to the primary care veterinarian was made to decrease the cyclosporine dose. Prior to dose reduction, a peak (2 hr after morning drug administration) cyclosporine blood concentration was measured through the Auburn University Clinical Pharmacology Laboratory to rule out an error in drug formulation, because the cyclosporine preparation had been compounded. The blood cyclosporine concentration was 1183 ng/mL. Although the cyclosporine concentration was within the Auburn Clinical Pharmacology Laboratory target therapeutic range for peak drug concentrations (400–1400 ng/mL), it was significantly lower, by a factor of two- to five-fold, than the peak drug concentration associated with high levels of T-cell suppression of IL-2 in pharmacodynamic studies in normal dogs.¹⁰

Because of the patient’s high degree of T-cell sensitivity to cyclosporine, the MDR1 gene mutation was considered to be a potential cause of heightened drug sensitivity. The MDR1 gene, also known as ATP-binding cassette sub family B member 1 (ABCB1) or permeability glycoprotein (P-glycoprotein) gene, encodes an efflux pump, which functions as a drug transporter for numerous agents, including cyclosporine.¹¹ Prevalence of MDR1 gene mutation (ABCB1-1Δ) is known to be higher in certain breeds, especially those of herding heritage, with the collie having the highest frequency.¹² The mutation has been described in the border collie as well, although prevalence is much less frequent compared with other herding breeds.¹² Dogs that are homozygous for the mutation have no P-glycoprotein function owing to a stop codon that ends synthesis of the protein prematurely, rendering it nonfunctional.¹³ Heterozygotes are also affected by the mutation and exhibit reduced P-glycoprotein function. A blood sample was sent to the Washington State University Veterinary Clinical Pharmacology Laboratory for DNA analysis, and the patient was found to be a heterozygote (mutant/normal) for the MDR1 gene mutation.

Subsequent samples submitted to the MSU Pharmacodynamic Laboratory after a reduction in cyclosporine dose rates demonstrated a significant decrease in T-cell suppression compared with initial qRT-PCR assay results. A second assay performed 8 wk after a marked cyclosporine dosage reduction (to 0.95 mg/kg PO q 12 hr) showed low levels of T-cell suppression. Because the anemia was not under control at this point (hematocrit 30%), the cyclosporine dosage was increased to 1.8 mg/kg PO q 12 hr. On repeat of the assay a few weeks later, qRT-PCR results again were reflective of low suppression, although anemia was adequately controlled (hematocrit 41%) and the owners reported that the patient was back to normal activity, with resolution of previous draining lesions. Because the patient appeared to be doing well clinically, a dose change was not recommended. A chronological diagram of the patient’s cyclosporine dose and IL-2 mRNA expression results over the course of her treatment is summarized in Figure 1.

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