Negative interference of icteric serum on a bichromatic biuret total protein assay.

BACKGROUND: Bilirubin is stated to be a negative interferent in some biuret assays and thus could contribute to pseudohypoproteinemia in icteric samples. OBJECTIVE: The purpose of the study was to evaluate the magnitude of and reason for a falsely low total protein concentration in icteric serum when the protein concentration is measured with a bichromatic spectrophotometric biuret assay. METHODS: Commercially available bilirubin was dissolved in 0.1 M NaOH and mixed with sera from 2 dogs to achieve various bilirubin concentrations of up to 40 mg/dL (first set of samples) and 35 mg/dL (second set of samples, for confirmation of first set of results and to explore the interference). Biuret total protein and bilirubin concentrations were determined with a chemistry analyzer (Cobas 6000 with c501 module). Line graphs were drawn to illustrate the effects of increasing bilirubin concentrations on the total protein concentrations. Specific spectrophotometric absorbance readings were examined to identify the reason for the negative interference. RESULTS: High bilirubin concentrations created a negative interference in the Cobas biuret assay. The detectable interference occurred with a spiked bilirubin concentration of 10.7 mg/dL in one set of samples, 20.8 mg/dL in a second set. The interference was due to a greater secondary-absorbance reading at the second measuring point in the samples spiked with bilirubin, which possibly had converted to biliverdin. CONCLUSION: Marked hyperbilirubinemia is associated with a falsely low serum total protein concentration when measured with a bichromatic spectrophotometric biuret assay. This can result in pseudohypoproteinemia and pseudohypoglobulinemia in icteric serum.

Refractometric total protein concentrations in icteric serum from dogs.

OBJECTIVE: To determine whether high serum bilirubin concentrations interfere with the measurement of serum total protein concentration by refractometry and to assess potential biases among refractometer measurements. DESIGN: Evaluation study. SAMPLE: Sera from 2 healthy Greyhounds. PROCEDURES: Bilirubin was dissolved in 0.1M NaOH, and the resulting solution was mixed with sera from 2 dogs from which food had been withheld to achieve various bilirubin concentrations up to 40 mg/dL. Refractometric total protein concentrations were estimated with 3 clinical refractometers. A biochemical analyzer was used to measure biuret assay-based total protein and bilirubin concentrations with spectrophotometric assays. RESULTS: No interference with refractometric measurement of total protein concentrations was detected with bilirubin concentrations up to 41.5 mg/dL. Biases in refractometric total protein concentrations were detected and were related to the conversion of refractive index values to total protein concentrations. CONCLUSIONS AND CLINICAL RELEVANCE: Hyperbilirubinemia did not interfere with the refractometric estimation of serum total protein concentration. The agreement among total protein concentrations estimated by 3 refractometers was dependent on the method of conversion of refractive index to total protein concentration and was independent of hyperbilirubinemia.

Acute megakaryoblastic leukemia in a German Shepherd dog.

An 11-year-old spayed-female German Shepherd dog was presented to the Veterinary Medical Teaching Hospital at Kansas State University with a history of weight loss, anorexia, depression, and lethargy for 2-3 weeks. Radiographic examination revealed a mass in the spleen and several round radiodense foci in the liver. CBC results included normocytic normochromic anemia, marked thrombocytopenia, and low numbers of neoplastic cells that frequently had cytoplasmic projections or blebs. A bone marrow aspirate contained about 80% neoplastic megakaryoblasts with the same microscopic features as those observed in peripheral blood. Using flow cytometry, cells of large size were identified in peripheral blood that expressed CD41/61, CD45, CD61, and CD62P (P-selectin) and were negative for markers of T cells, B cells, monocyte/macrophages, and dendritic cells. Because of the poor prognosis, euthanasia and subsequently necropsy were performed. On histopathologic examination, neoplastic megakaryoblasts were identified in spleen, liver, mesenteric lymph node, and the pulmonary vasculature. Using immunohistochemistry, the neoplastic megakaryoblasts weakly expressed von Willebrand factor. Based on microscopic and immunophenotypic findings, a diagnosis of acute megakaryoblastic leukemia (AMegL) was made. To our knowledge, this is the first report of AMegL in a domestic animal in which immunophenotyping by flow cytometry and a panel of antibodies against CD41/61, CD61, and CD62P were used to support the diagnosis.

Canine elliptocytosis due to a mutant beta-spectrin.

A 5-year-old, spayed female, mixed-breed dog with persistent elliptocytosis was evaluated at the Veterinary Medical Teaching Hospital at Kansas State University. The elliptocytosis was asymptomatic and was detected during the evaluation of lameness. When subjected to shear stress in an ektacytometer, the dog's erythrocytes had reduced cellular deformability and erythrocyte membranes had decreased mechanical stability. Analysis of erythrocyte membrane spectrin by nondenaturing gel electrophoresis revealed an increased amount of spectrin dimers, indicating a defect in spectrin self-association. DNA analysis detected a beta-spectrin mutation in codon 2110 in which threonine was replaced by methionine. This mutation likely altered the molecular structure of the erythrocyte membrane, leading to impaired spectrin self-association and elliptocyte formation.

What is your diagnosis? Muculent pleural effusion from a dog.

Pleural effusion was examined from a 5-year-old, female Brittany Spaniel with a 7-day history of dyspnea, anorexia, and diarrhea. The fluid was yellow, cloudy, and slightly gelatinous, and had a total protein concentration of 2.8 g/dL, a total nucleated cell concentration of 1.1 x 10(3)/microL, and a triglyceride concentration of 177 mg/dL. A cytocentrifuged preparation contained a mixed inflammatory cell population with a predominance of small lymphocytes and abundant mucinous material in the background. The dog died 3 days later and a mass was found within the lumen and wall of the right auricle of the heart at necropsy. Histopathologic sections of the mass contained a population of anaplastic spindle cells diffusely suspended in a pale basophilic matrix, consistent with myxosarcoma. The cells were positive for vimentin and negative for cytokeratin, desmin, and von Willebrand factor VIII-related antigen. A myxoid matrix was confirmed by positive staining with Alcian blue. Myxosarcoma is a rare cardiac tumor in dogs that should be considered, along with mucus-producing carcinomas and bile, as a cause of muculent effusion.

Blood smear from a cat: features to "dys"cover.

A 4-year-old, spayed female, domestic shorthair cat was presented for lethargy, nonregenerative anemia, and inappetence. Results of a CBC included macrocytic, normochromic, nonregenerative anemia and a glucocorticoid-associated leukogram. On blood smear examination, neutrophils had abnormal features including hyposegmentation and a diffuse chromatin pattern with nuclear filament formation and nuclear blebbing. Microscopic examination of a roll preparation of bone marrow revealed hypolobulated megakaryocytes with asynchronous maturation of nuclei. The granulocytic to erythrocyte (G:E) ratio was 76. Segmented neutrophils had asynchronous maturation and dysplastic features. The entire erythroid lineage was markedly decreased for the degree of anemia and rare dysplastic features were noted in erythroid precursor cells. The interpretation of bone marrow findings was erythroid hypoplasia, megakaryocytic dysplasia, and granulocytic hyperplasia with dysplasia. Histopathologic examination of a bone marrow core sample also revealed myeloid hyperplasia and erythroid hypoplasia. The result of a direct immunofluorescence assay for FeLV performed on the bone marrow roll preparation was positive. A diagnosis of dysmyelopoiesis associated with FeLV infection was made. This case was unique in that the dysplastic changes occurred in cell lines that did not have associated cytopenias. The dysmyelopoiesis most closely resembled myelodysplastic syndrome with refractory cytopenia (MDS-RC); however, secondary dysmyelopoiesis could not be ruled out.

Clinical assessment of leukocytosis: distinguishing leukocytoses caused by inflammatory, glucocorticoid, physiologic, and leukemic disorders or conditions.

The four major types of leukocytoses are inflammatory, glucocorticoid-associated, catecholamine-associated, and neoplastic. These leukocytoses are distinguished by leukocyte concentrations, microscopic features of leukocytes, and associations with other laboratory data. All laboratory findings need to be interpreted within the context of the case information, including signalment, history, and physical examination findings. Newer assays are being used to differentiate the different forms of leukocyte neoplasia and to distinguish between hyperplastic and neoplastic proliferations.

Predominance of Ehrlichia ewingii in Missouri dogs.

To investigate the species distribution of Ehrlichia present in Missouri dogs, we tested 78 dogs suspected of having acute ehrlichiosis and 10 healthy dogs. Blood from each dog was screened with a broad-range 16S rRNA gene PCR assay that detects known pathogenic species of Ehrlichia and ANAPLASMA: The species was determined by using species-specific PCR assays and nucleotide sequencing. Ehrlichia antibody testing was performed by using an indirect immunofluorescence assay with Ehrlichia chaffeensis as the antigenic substrate. The broad-range assay detected Ehrlichia or Anaplasma DNA in 20 (26%) of the symptomatic dogs and 2 (20%) of the asymptomatic dogs. E. ewingii accounted for 20 (91%), and E. chaffeensis accounted for 1 (5%) of the positives. Anaplasma phagocytophilum DNA was detected in one dog, and the sequences of regions of the 16S rRNA gene and the groESL operon amplified from the blood of this dog matched the published sequences of this organism. Antibodies reactive with E. chaffeensis were detected in 14 (67%) of the 21 PCR-positive dogs and in 12 (19%) of the 64 PCR-negative dogs. Combining the results of PCR and serology indicated that 33 (39%) of 85 evaluable dogs had evidence of past or current Ehrlichia infection. We conclude that E. ewingii is the predominant etiologic agent of canine ehrlichiosis in the areas of Missouri included in this survey. E. canis, a widely recognized agent of canine ehrlichiosis, was not detected in any animal. The finding of E. ewingii in asymptomatic dogs suggests that dogs could be a reservoir for this Ehrlichia species.

Blood values in wild and captive Komodo dragons (Varanus komodoensis).

The Komodo dragon (Varanus komodoensis) is the largest living lizard and occupies a range smaller than that of any other large carnivore in the world. Samples from 33 free-ranging animals at five localities in Komodo National Park, Indonesia were evaluated to assess underlying health problems. To build a comparative database, samples from 44 Komodo dragons in both Indonesian and U.S. zoos were also analyzed. Tests performed included complete blood counts, clinical chemistry profiles, vitamin A, D(3), and E analyses, mineral levels, and screening for chlorinated pesticides or other toxins in wild specimens. Blood samples from wild dragons were positive for hemogregarines, whereas captive specimens were all negative. Total white blood cell counts were consistently higher in captive Komodo dragons than in wild specimens. Reference intervals were established for some chemistry analytes, and values obtained from different groups were compared. Vitamin A and E ranges were established. Vitamin D(3) levels were significantly different in Komodo dragons kept in captive, indoor exhibits versus those with daily ultraviolet-B exposure, whether captive or wild specimens. Corrective measures such as ultraviolet-permeable skylights, direct sunlight exposure, and self-ballasted mercury vapor ultraviolet lamps increased vitamin D(3) concentrations in four dragons to levels comparable with wild specimens. Toxicology results were negative except for background-level chlorinated pesticide residues. The results indicate no notable medical, nutritional, or toxic problems in the wild Komodo dragon population. Problems in captive specimens may relate to, and can be corrected by, husbandry measures such as regular ultraviolet-B exposure. Zoo Biol 19:495-509, 2000. Copyright 2000 Wiley-Liss, Inc.

Analysis of serum protein concentrations after severe thermal injury in a dog.

Serial serum electrophoreses and routine serum protein assays were used to assess changes in serum protein concentrations after severe thermal injury in a dog. Electrophoretic patterns during the month of evaluation were consistent with protein-losing dermatopathy (thermal burn) and inflammatory dysproteinemias.