To validate the use of a flow cytometric assay that uses 2‘,7‘-dichlorodihydrofluorescein diacetate (DCFH-DA) to measure reactive oxygen species in the erythrocytes of healthy dogs.
50 healthy adult dogs.
Erythrocytes were incubated with DCFH-DA or a vehicle control (dimethyl sulfoxide), then incubated with (stimulated) or without (unstimulated) hydrogen peroxide. The flow cytometric assay was evaluated for specificity with increasing concentrations of DCFH-DA and hydrogen peroxide, and a polynomial regression line was applied to determine optimal concentrations. For precision, samples were analyzed 5 consecutive times for determination of intra- and interassay variability. Stability of samples stored at 4°C for up to 48 hours after blood collection was determined with flow cytometric analysis. Coefficient of variation (CV) was considered acceptable at 20%. Baseline measurements were used to determine an expected range of median fluorescence intensity for unstimulated erythrocytes incubated with DCFH-DA.
Erythrocytes were successfully isolated, and stimulated samples demonstrated higher median fluorescence intensity, compared with unstimulated samples. The intra-assay CV was 11.9% and 8.9% and interassay CV was 11.9% and 9.1% for unstimulated and stimulated samples, respectively. Unstimulated samples were stable for up to 24 hours, whereas stimulated samples were stable for up to 48 hours.
CONCLUSIONS AND CLINICAL RELEVANCE
Flow cytometry for the measurement of reactive oxygen species in the erythrocytes of healthy dogs by use of DCFH-DA had acceptable specificity, precision, and stability. Flow cytometry is a promising technique for evaluating intraerythrocytic oxidative stress for healthy dogs.
Objective—To evaluate sensitivity and specificity of a
real-time reverse transcriptase polymerase chain
reaction (RT-PCR) assay performed on pooled nasal
swab specimens, compared with virus isolation performed
on individual nasal swab specimens by use of
2 cell culture lines for detection of swine influenza A
Sample Population—900 nasal swab specimens
obtained from pigs at an abattoir and 62 nasal swab
specimens submitted for diagnostic testing.
Procedure—Primers were chosen to amplify a conserved
portion of the influenza virus matrix gene.
Assay sensitivity was initially determined by testing
serial dilutions of various subtypes of swine influenza
viruses. Sensitivity and specificity were confirmed by
use of nasal swab specimens with or without addition
of known concentrations of influenza virus and further
validated by testing nasal swab specimens obtained
through an abattoir surveillance program or submitted
for diagnostic testing. Aliquots of specimens were
pooled in sets of 10, and results of real-time RT-PCR
assays were compared with results of virus isolation
of individual specimens in Madin Darby canine kidney
(MDCK) and mink lung (Mv1Lu) cells.
Results—Real-time RT-PCR assay was highly specific
(100%) and sensitive (88% to 100%). Among the 16
viruses isolated, 3 grew only in Mv1Lu cells and 3
grew only in MDCK cells.
Conclusions and Clinical Relevance—Results indicate
that real-time RT-PCR assay is a fast and accurate
test for screening numerous nasal swab specimens for
swine influenza virus. Some viruses were isolated in
only MDCK or Mv1Lu cells, indicating that use of
> 1 cell line may be required to isolate a broad range of
influenza A viruses. (Am J Vet Res 2005;66:119–124)
Objective—To evaluate the effects of nephrotomy on
renal function in clinically normal cats.
Animals—20 specific-pathogen-free, 9- to 11-month old
female mixed-breed cats.
Procedure—Serum chemistry analyses, CBC determinations,
urinalyses, microbiologic urine cultures,
renal ultrasonography, abdominal radiography, and
single-kidney and total glomerular filtration rate (GFR)
determinations by use of renal scintigraphy and measurements
of plasma disappearance of technetium
99m-diethylenetriaminepentaacetic acid were performed
before surgery and at 3, 12, 26, 52, and 78
weeks after surgery in 10 cats that underwent unilateral
nephrotomy and in 10 control cats that underwent
a sham surgical procedure.
Results—Two cats (1 from each group) did not complete
the study, and their data were eliminated from
analyses. Unilateral nephrotomy resulted in a 10% to
20% reduction in mean single-kidney GFR, compared
with that of nephrotomy contralateral control kidneys.
However, mean total GFR in nephrotomy-group cats
was not significantly different from that of shamgroup
cats. Over the 78 weeks of study, mean total
GFR declined 34% and 40% in nephrotomy- and
sham-group cats, respectively. Adverse events associated
with nephrotomy included persistent microscopic
hematuria, renal pelvis hyperechogenicity with
distant shadowing on ultrasonographic evaluation,
dilatation of renal pelves, and hydronephrosis.
Conclusions and Clinical Relevance—Nephrotomy
in normal functioning feline kidneys results in a modest
relative reduction in renal function, compared with
contralateral kidney controls, but has minimal effect
on total GFR when compared with sham-operated
control cats. However, any detrimental effects of
nephrotomy may be magnified in cats with diseased
kidneys, which may have little or no capacity for repair
or compensation. (Am J Vet Res 2005;66:1400–1407)
Case Description—An 11-year-old 72-kg (158-lb) sexually intact female alpaca was examined for diagnosis and treatment of hematuria of 4 months' duration.
Clinical Findings—Pigmenturia was detected by the owner when the alpaca was 8 months pregnant. Radiographic, ultrasonographic, vaginal speculum, and cystoscopic evaluation of the urinary tract revealed normal vaginal and urethral epithelia and increased bladder vessel tortuosity, with pulses of hemorrhage from the left ureter. Regenerative anemia and mild leukopenia were detected and serum urea nitrogen and creatinine concentrations were within reference ranges.
Treatment and Outcome—Chronic hematuria resolved after unilateral nephrectomy of the left kidney, and no dysfunction was detected in the remaining kidney. Histologic evaluation of the kidney revealed a transitional cell tumor in the renal pelvis.
Clinical Relevance—Although anemia is common in South American camelids, hematuria is an uncommon sign of this condition. Chronic urinary tract infection, toxin ingestion, and neoplasia causing hematuria or hemoglobinuria should be considered in South American camelids with pigmenturia. Thorough and systematic evaluation of the urinary tract should be performed to locate the site of hemorrhage to treat hematuria appropriately.