Detection of antiplatelet antibody: comparison of platelet immunofluorescence,agglutination, and immunoinjury tests using rabbit antiequine platelet serum

Immunofluorescence, tube agglutination, and platelet factor-3 immunoinjury tests for detecting antiplatelet antibody were compared using a heterologous system of equine platelets and rabbit antiequine platelet serum. Platelet immunofluorescence tests were performed using paratormaldehyde-fixed platelets in suspension as well as in air-dried smears on glass slides (solid phase). Bright homogeneous, membranous, specific fluorescence was seen in both assays with anti-immunoglobulin G (IgG) and protein G fluorescein isothiocynate conjugates (FITC-conjugates). Protein A conjugate gave nonspecific fluorescence irrespective of normal or antiserum treatment. Anti-IgG and protein G conjugates in suspension immunofluorescence tests with the same antiserum yielded antibody titers of 1:1024 and 1:128, respectively. Similarly, respective titers of 1:512 and 1:64 were obtained with solid phase immunoassay. Platelet suspension assay was slightly better than the solid phase assay. These observations indicated that anti-IgG was more sensitive than protein G in detecting antiplatelet antibody by fluorescence microscopy, while protein A was ineffective because of its nonspecificity. Chloroquine treatment of platelets failed to reduce the nonspecific fluorescence. Platelet agglutination and platelet factor-3 tests were relatively less sensitive to detect equine antiplatelet antibody.     

Monoclonal Gammopathy Associated With Naturally Occurring Canine Ehrlichiosis

Clinical, hematologic, and immunologic findings for 14 dogs with Ehrlichia canis monoclonal gammopathy were studied retrospectively. Epistaxis, anemia, thrombocytopenia, hypoalbuminemia, hypergammaglobulinemia, and proteinuria were documented in the majority of these dogs. The serum protein electrophoresis pattern was characterized by a distinct narrow-base monoclonal spike, by a broad-base monoclonal spike, or by a monoclonal spike superimposed on a polyclonal gammopathy. The monoclonal spike disappeared following tetracycline treatment for ehrlichiosis. The long-term prognosis following treatment was generally good. The diagnostic features of monoclonal gammopathy due to myeloma were compared with those of E. canis monoclonal gammopathy. Owing to numerous similarities in clinical, hematologic, and immunologic findings, we conclude that an E. canis antibody titer should be determined in all dogs in which a diagnosis of benign monoclonal gammopathy is contemplated or definitive evidence of myeloma, leukemia, or macroglobulinemia is lacking.     

Comparison of Multiplate,Platelet Function Analyzer‐200, and Plateletworks in Healthy Dogs Treated with Aspirin and Clopidogrel

Background

Platelet function testing may be warranted to assess response to aspirin and clopidogrel.

Hypothesis/Objectives

To evaluate the effects of aspirin, clopidogrel, or combination therapy using 3 platelet function tests: Multiplate Analyzer (MP), Platelet Function Analyzer‐200 (PFA), and Plateletworks (PW).

Animals

Six healthy laboratory Beagles.

Methods

Randomized double‐blind placebo‐controlled study (crossover design). Dogs were given aspirin 1 mg/kg, clopidogrel 2 mg/kg, or combination therapy for 1 week each, with a washout period of 2 weeks. Platelet function was assessed on days 0 and 7 of each phase using MP (adenosine diphosphate [ADP], arachidonic acid [AA], collagen [COL] agonists), PFA (P2Y, COL‐ADP [CADP], COL‐Epinephrine [CEPI] cartridges), and PW (ADP, AA, COL agonists). Platelet counts were obtained with impedance and optical counters.

Results

For MP, mean aggregation was decreased for COL and AA with combination therapy and for ADP with all treatments. For PFA, mean CT was increased for the CEPI cartridge with aspirin; and for the P2Y and CADP cartridges with clopidogrel or combination therapy. More dogs receiving clopidogrel showed an increase in PFA CT using the P2Y than the CADP cartridge. For PW, mean aggregation was decreased for AA with all treatments; for ADP with clopidogrel or combination therapy; and for COL with clopidogrel. The PW results with the 2 hematology counters showed almost perfect agreement.

Conclusion and Clinical Importance

All platelet function tests detected treatment effects in some dogs and may have utility for monitoring therapy.     

Monoclonal gammopathy in a dog with plasmacytic gastroenterocolitis.

Monoclonal gammopathy associated with plasmacytic gastroenterocolitis was diagnosed in a dog. Treatment consisted of immunosuppressive drugs and dietary manipulation. The gammopathy resolved. Fifteen months after initial diagnosis, the dog had a relapse of intestinal disease and recurrence of the gammopathy. After inducing a second remission, the gammopathy again resolved.     

Biclonal gammopathy associated with immunoglobulin A in a dog with multiple myeloma

A 10-year-old neutered male Airedale Terrier was evaluated for inappetance, weight loss, and lameness. Multiple myeloma was diagnosed based on bone marrow plasmacytosis, multiple lytic bone lesions, and hyperglobulinemia with a clonal gammopathy on serum protein electrophoresis. Splenic plasmacytosis, and retinal lesions consistent with hyperviscosity syndrome also were found. Temporary responses to 2 different chemotherapy protocols (melphalan and prednisone, and cyclophosphamide and prednisone) were seen, with remission of clinical signs and a decrease in the biclonal gammopathy but no resolution of the splenic mass. Eventual return of clinical signs led to euthanasia at 175 days postdiagnosis. Necropsy examination confirmed multiple myeloma involving bone marrow and spleen, and glomerulonephritis. An immunoglobulin-A (IgA) gammopathy was demonstrated by immunoelectrophoresis; biclonality was ascertained by immunofixation electrophoresis. The clonal components consisted of intact Ig with a heavy chain of the a class and a light chain of undetermined class. To our knowledge, this is the first report of undimerized biclonal gammopathy in a dog caused by a single heavy chain class involving IgA.     

Progression of a solitary, malignant cutaneous plasma-cell tumour to multiple myeloma in a cat

An 11‐year‐old male domestic shorthair cat was examined because of a soft‐tissue mass on the left tarsus previously diagnosed as a malignant extramedullary plasmacytoma. Findings of further diagnostic tests carried out to evaluate the patient for multiple myeloma were negative. Five months later, the cat developed clinical evidence of multiple myeloma based on positive Bence Jones proteinuria, monoclonal gammopathy and circulating atypical plasma cells. This case represents an unusual presentation for this disease and documents progression of an extramedullary plasmacytoma to multiple myeloma in the cat.     

Monoclonal Gammopathy in a Dog With Chronic Pyoderma

A monoclonal gammopathy composed of immunoglobulin G, with concurrent light-chain proteinuria and generalized lymph node plasmacytosis, was associated with chronic pyoderma in a dog. A uniform population of plasma cells was observed cytologically and histologically in multiple lymph node specimens. A diagnosis of monoclonal gammopathy of unknown significance was eventually made by exclusion of other known causes of monoclonal gammopathy, resolution after antibiotic therapy, and no evidence of lymphoproliferative disease after 11 months of follow-up and subsequent necropsy. This report expands the diagnostic considerations for monoclonal gammopathies in the dog.     

Haemostatic mechanisms of the newborn foal: Reduced platelet responsiveness

Whole blood platelet counts, coagulation profiles and in vitro platelet function tests were monitored in newborn foals during the first week of life. Platelet counts, mean platelet volumes and thrombin-induced malondialdehyde production were not different from adult mares. Prothrombin and partial thromboplastin times were slightly, but not significantly, longer for neonatal blood samples than for mare samples. Platelet aggregation responses to serotonin, arachidonic acid or adrenaline did not change during the study. On the other hand, adenosine diphosphate-induced aggregation and collagen-induced aggregation increased progressively over the first week of life. Adrenaline exposure diminished adenosine diphosphate-induced aggregation only during the first 12 h of life. The results of this study indicate that the haemostatic mechanisms of equine neonates are immature at birth and that, during the maturation period, the equine neonate may be at risk of platelet-associated haemorrhagic disorders.     

Multiple myeloma in a dog with multiple concurrent infectious diseases and persistent polyclonal gammopathy

A 12‐year‐old, spayed female, mixed‐breed dog was presented for acute hematuria, stranguria, polyuria, and polydipsia, as well as lameness for 8 days. Previous medical history included treatment for infection with Ehrlichia canis, Anaplasma phagocytophilum, Leishmania infantum, and Dirofilaria immitis 6.5 years prior to presentation. Besides persistently increased antibody titers to E canis and A phagocytophilum, polyclonal gammopathy with a monoclonal spike and moderate hypercalcemia were observed. There was marked hematuria, and Staphylococcus aureus was cultured from urine. Two weeks after successful treatment of the urinary tract infection, radiographs showed an extensive destructive monostotic lesion of the right humerus. Cytologic examination of fine‐needle aspirates of this lesion revealed a neoplastic round cell population suggestive of multiple myeloma. The dog was treated with melphalan and prednisolone for suspected multiple myeloma and doxycycline for suspected ehrlichiosis and anaplasmosis. Treatments lead to resolution of the clinical signs, hypercalcemia, and monoclonal gammopathy, and there was radiographic improvement of bone lesions; polyclonal gammopathy persisted. About one year after presentation the dog was still in clinical remission. This is a rare report of a dog with suspected multiple myeloma and a history of multiple chronic infectious diseases, suggesting that chronic infection and uncontrolled long‐term stimulation of the immune system could contribute to the pathogenesis of multiple myeloma.     

Immunoglobulin A monoclonal gammopathy in two horses with multiple myeloma

The clinical findings in two horses with secretory multiple myeloma and secondary immunoglobulin A (IgA) monoclonal gammopathy were non-specific and included weight loss, pale mucous membranes, limb oedema and bacterial respiratory tract infection. Consistent laboratory abnormalities included hyperproteinaemia, hyperglobulinaemia, hypoalbuminaemia and hypercalcaemia. The diagnosis was based on the presence of IgA monoclonal gammopathy in serum and urine and bone marrow plasmacytosis (> 10 per cent). One horse was euthanased; it had neoplastic plasma cell infiltrates in its kidneys, spleen, liver, bone marrow, myocardium and adrenal glands. The other horse was treated for a bacterial pneumonia and was still alive six months after it was first examined.     

Characterization of the equine platelet aggregation response.

Equine platelet aggregation responses to bovine collagen, adenosine diphosphate (ADP), serotonin, epinephrine, and arachidonate in a platelet aggregometer were recorded. Equine platelets exhibited irreversible aggregation when incubated with ADP at a final concentration of 10 microM and bovine collagen. A secondary aggregation wave was recorded from platelets from certain horses at final ADP concentrations of 1 to 5 microM. Serotonin and arachidonate induced a weak reversible aggregation response, but a response was not observed following epinephrine addition. Equine platelet aggregation was influenced by concentration of anticoagulant (sodium citrate). Platelet aggregation responses at 37 C were indistinguishable from those recorded at 39 C. Platelet aggregation responses also were altered if the aggregation tests were not performed within 4 hours of blood sample acquisition. An assessment of platelet aggregation from multiple blood samples from the same horse indicated that the procedures described provide a reliable method to assess equine platelet aggregation in vitro.     

Monoclonal Gammopathy in a Dog With Visceral Leishmaniasis

One dog with visceral leishmaniasis associated with monoclonal gammopathy is described. Most dogs with visceral leishmaniasis present with hyperproteinemia due to a polyclonal gammopathy, but the possibility of monoclonal gammopathy must be considered. Because dogs accompany their owners when they travel, the diagnosis of leishmaniasis should be considered if an animal with monoclonal gammopathy has visited an area where the disease is endemic. The observation of Leishmania in the macrophages of a bone marrow, lymph node smear, or skin biopsy specimen is diagnostic.     

Spurious hyperphosphatemia in a dog with chronic lymphocytic leukemia and an IgM monoclonal gammopathy

Spurious hyperphosphatemia was diagnosed in a 6-year-old, neutered female, mixed-breed dog with chronic lymphocytic leukemia associated with an IgM monoclonal gammopathy. The spurious hyperphosphatemia was probably caused by paraprotein precipitation which interfered with the ASTRA 8 automated analyzer measurements. Serial dilutions of the sample did not change the phosphorus value. Another analyzer system in which a protein-free sample was prepared prior to analysis gave a normal serum phosphorus concentration. There was a linear relationship between the amount of paraprotein and the measured total serum inorganic phosphate (r=0.75). A review of 700 chemistry profiles from dogs and cats and a review of 36 cases with polygonal gammopathy and 6 cases with monoclonal gammopathy did not reveal other cases of spurious hyperphosphatemia.     

A metastatic secretory gastric plasmacytoma with aberrant CD3 expression in a dog

A 10‐year‐old crossbred dog was presented with a 6‐week history of hematemesis, melena, anorexia, and lethargy. Clinical evaluation revealed a gastric mass with a regional lymphadenomegaly as well as a monoclonal gammopathy manifesting as hyperglobulinemia. Cytologic and histopathologic analyses were consistent with a round cell neoplasm; neoplastic cells showed nuclear immunoreactivity for MUM1 and diffuse cytoplasmic reactivity for CD3. Polymerase chain reactions performed on fixed and fresh tissue identified a clonal rearrangement with an IgH primer set. An extramedullary plasmacytoma (EMP) was confirmed by cellular morphology and molecular diagnostics. Following an objective response to chemotherapy, the dog was euthanized 8 months after diagnosis, and a postmortem examination confirmed the clinical findings. This is the first reported case of a monoclonal gammopathy secondary to a gastric EMP coupled with aberrant expression of CD3 in an aggressive plasmacytic tumor, and highlights the utility of molecular diagnostics for classifying atypical hemolymphoid neoplasms.     

Use of plasmapheresis and chemotherapy for treatment of monoclonal gammopathy associated with Ehrlichia canis infection in a dog

Monoclonal gammopathy associated with Ehrlichia canis infection was diagnosed in a German Shepherd Dog. The dog was treated by use of chemotherapy and tetracycline and by plasmapheresis. The dog tolerated plasmapheresis and long-term drug therapy well, and clinical signs resolved over a 90-day period. The monoclonal gammopathy resolved after treatment, but specific antibody to E canis indicated suppression followed by a rebound to the initial high titer.     

A primary hepatic plasma cell tumor in a dog

An 8-year-old female Shetland sheep dog had hyperproteinemia with a monoclonal gammopathy and a solid mass on the liver, which was histologically diagnosed as a plasma cell tumor. After the treatment of surgery and chemotherapy, serum protein level reduced to the normal range and the gammopathy was disappeared. These findings indicate the plasma cell tumor developed primarily from the liver.     

Elevated fructosamine concentrations caused by IgA paraproteinemia in two dogs

An 8-year-old male Austrian Pinscher and a 14-year-old male Golden Retriever were presented for evaluation due to unexplainable high fructosamine values despite euglycemia and epistaxis in combination with polydipsia/polyuria, respectively. Blood analysis revealed severe hyperglobulinemia, hypoalbuminemia and markedly elevated fructosamine concentrations in both dogs. Multiple myeloma with IgA-monoclonal gammopathy was diagnosed by serum and urine electrophoresis including immunodetection with an anti-dog IgA antibody and bone marrow aspirations. Diabetes mellitus was excluded by repeated plasma and urine glucose measurements. Fructosamine values were positively correlated with globulin, but negatively correlated with albumin concentrations. These cases suggest that, as in human patients, monoclonal IgA gammopathy should be considered as a possible differential diagnosis for dogs with high fructosamine concentrations.     

Gammopathy in a Spanish dog infected with Bartonella henselae

Generalised pyogranulomatous disease and hyperviscosity syndrome associated with a presumed monoclonal gammopathy was diagnosed in a three-year-old intact female Pomeranian. The Bartonella henselae antibody titer was 1:64 and Bartonella species DNA was amplified from the splenic tissue. Monoclonal gammopathies in dogs are typically associated with plasma cell and lymphoid dyscrasias and other inflammatory or infectious diseases such as ehrlichiosis and leishmaniosis. Based on this case report, infection with Bartonella species should also be added to the differential diagnoses for gammopathy in dogs. To the authors' knowledge, this is the first report of molecular evidence of Bartonella species infection in a sick dog in Spain.     

Unusual IgM-secreting multiple myeloma in a dog

A 4-year-old castrated male dog was evaluated because of multiple-limb lameness. Signs of pain were elicited during palpation of the regions of the proximal tibial metaphyses and distal left radial diaphysis. Radiography revealed osteolytic lesions of the long bones. Blood analyses revealed hypercalcemia and transient cytopenias. Serum protein electrophoresis did not reveal a monoclonal gammopathy; however, urine protein electrophoresis revealed Bence Jones proteinuria. Serial diagnostic sampling of bone lesions, immunohistochemical staining methods, and serum and urine protein immunoelectrophoresis were required to establish a diagnosis of multiple myeloma. Two IgM components were identified via serum protein immunoelectrofixation. The dog improved clinically after initiation of chemotherapy with melphalan and prednisone; however, the dog ultimately was euthanatized because of pathologic fracture. The case was unique because there was lack of vertebral involvement, an unusual gammopathy, and difficulty in identifying myeloma cells via serial sampling.     

Inhibition of platelet function with clopidogrel,as measured with a novel whole blood impedance aggregometer in horses

This study aimed to validate a loading and maintenance clopidogrel dosing scheme for the inhibition of platelet function, measured by whole blood impedance aggregometry in healthy adult horses. Ten Warmblood horses received oral clopidogrel once daily. Doses were based on 50 kg weight categories and resulted in one loading dose of 6–6.5 mg/kg bodyweight and maintenance doses of 1.2–1.4 mg/kg over the next 4 days. Platelet function was measured via whole blood multiple electrode impedance aggregometry prior to (T0) and at 6, 12, 24, 48, 72, 96, 144, 192 and 240 h following the loading dose. Aggregometries for collagen (COLtest), arachidonic acid (ASPItest), adenosine diphosphate (ADPtest) and ADP with prostaglandin E1 (ADPtestHS) were performed. Statistical analyses included one way repeated measures ANOVAs and subsequent Dunnett's tests.Platelet aggregation induced by collagen remained unchanged. There were significant inhibitions in the ASPItest (P <0.01 at 192 h, and P <0.05 at 240 h) and the ADPtest and ADPtestHS (P < 0.01, with the exception of 240 h). The loading dose of clopidogrel induced rapid inhibition of platelet function within hours, and the low dose was suitable for maintaining the inhibition over the 4 days of therapy. Recovery of platelet function was restored 6 days after the cessation of medication, determined with the ADPtest and ADPtestHS, but remained inhibited with the ASPItest. The prolonged effect of clopidogrel may indicate differences in the activation of platelets between horses and humans that were previously unknown.