scholarly journals Comparison of real-time reverse transcriptase polymerase chain reaction of peripheral blood mononuclear cells, serum and cell-free body cavity effusion for the diagnosis of feline infectious peritonitis

2016 ◽  
Vol 19 (4) ◽  
pp. 344-350 ◽  
Author(s):  
Stephanie J Doenges ◽  
Karin Weber ◽  
Roswitha Dorsch ◽  
Robert Fux ◽  
Katrin Hartmann
Keyword(s):  
Real Time ◽  
Mononuclear Cells ◽  
Body Cavity ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Peripheral Blood Mononuclear ◽  
Feline Infectious Peritonitis ◽  
Blood Mononuclear Cells ◽  
Polymerase Chain ◽  
Free Body

Objectives Diagnosis of feline infectious peritonitis (FIP) remains challenging, especially in cats without effusions. The objective of this study was to evaluate the sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction (RT-PCR) detecting feline coronavirus (FCoV) RNA in peripheral blood mononuclear cells (PBMCs) and serum in comparison with the same real-time RT-PCR in cell-free body cavity effusion. Methods This prospective case-control study included 92 cats. Forty-three cats had a definitive diagnosis of FIP, established either by histopathological examination (n = 28) or by positive immunofluorescence staining of FCoV antigen in macrophages of effusions (n = 11), or by both methods (n = 4). Forty-nine control cats had other diseases but similar clinical signs. Real-time RT-PCR was performed on PBMCs of 37 cats (21 cats with FIP, 16 controls), on serum of 51 cats (26 cats with FIP, 25 controls) and on cell-free body cavity effusion of 69 cats (36 cats with FIP, 33 controls). Sensitivity, specificity, positive and negative predictive value, including 95% confidence intervals (CI), were calculated. Results Real-time RT-PCR of PBMCs, serum and cell-free body cavity effusion showed a specificity of 100% (95% CI 79.4–100% in PBMCs, 86.3–100% in serum, 89.4–100% in cell-free body cavity effusion) and a sensitivity of 28.6% (95% CI 11.3–52.2%) in PBMCs, 15.4% (95% CI 4.4–34.9%) in serum and 88.9% (95% CI 73.9–96.9%) in cell-free body cavity effusion to diagnose FIP. Conclusions and relevance Although it is known that RT-PCR can often provide false-positive results in healthy cats, this real-time RT-PCR was shown to be a specific tool for the diagnosis of FIP when applied in a clinical setting. Sensitivity in cell-free body cavity effusion was high but low in PBMCs and serum. PBMC samples showed a higher sensitivity than serum samples, and are therefore a better choice if no effusion is present.

scholarly journals Quantification of hepatitis C virus-infected peripheral blood mononuclear cells by in situ reverse transcriptase-polymerase chain reaction

Blood ◽  
1996 ◽  
Vol 88 (7) ◽  
pp. 2768-2774 ◽  
Author(s):  
L Muratori ◽  
D Gibellini ◽  
M Lenzi ◽  
M Cataleta ◽  
P Muratori ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Chronic Hepatitis ◽  
Hepatitis C ◽  
Mononuclear Cells ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Polymerase Chain

Hepatitis C virus (HCV) is known to infect peripheral blood mononuclear cells (PBMC) of patients with chronic hepatitis C, but the proportion of HCV-infected circulating cells is not detectable by conventional reverse transcriptase-polymerase chain reaction (RT-PCR) and the pathogenic significance of HCV lymphotropism is still unclear. Therefore, we have devised an in situ RT-PCR technique using fluorescein-labeled HCV-specific primers revealed by flow cytometry. PBMC were isolated from 28 patients with chronic HCV-related liver disease; of these, 6 had previously received an orthotopic liver transplantation (OLT) and were on immuno-suppressive treatment. Fourteen patients (50%) were found positive for HCV genome within PBMC by in situ RT-PCR, the proportion of HCV-infected cells ranging from 0.2% to 8.1%. All 6 OLT patients tested positive. The fluorescent signal, corresponding to the HCV-specific 340-bp amplicon, was confined to part of the cytoplasmic compartment of scattered PBMC. Of these 14 patients, 12 had also negativestrand HCV RNA within PBMC detected by “tagged” RT-PCR. We conclude that HCV may infect a significant proportion of PBMC in chronic hepatitis C patients, especially immunosuppressed OLT cases, and that viral replication within PBMC is a common occurrence. Over time, the persistence of HCV-infected immune system cells might interfere with normal immunologic mechanisms and play a role in the pathogenic processes leading to extrahepatic disorders such as mixed cryoglobulinemia and B-cell malignant lymphoma.

Detection of tumor cells in purged bone marrow and peripheral-blood mononuclear cells by polymerase chain reaction amplification of bcl-2 translocations.

1994 ◽  
Vol 12 (5) ◽  
pp. 1021-1027 ◽  
Author(s):  
R S Negrin ◽  
J Pesando
Keyword(s):  
Bone Marrow ◽  
Polymerase Chain Reaction ◽  
Tumor Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Mononuclear Cells ◽  
Chain Reaction ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Before And After ◽  
Polymerase Chain

PURPOSE To compare bone marrow (BM) before and after purging with monoclonal antibodies (MAbs) and complement with peripheral-blood mononuclear cells (PBMNCs) for tumor-cell contamination by amplification of t(14;18) sequences using the polymerase chain reaction (PCR). PATIENTS AND METHODS Sixty patients with non-Hodgkin's lymphoma (NHL) undergoing autologous BM transplantation were evaluated. Six BM biopsies were performed at the time of harvesting and evaluated morphologically for tumor involvement. The harvested BM was treated with a panel of anti-B-cell MAbs directed against CD9, CD10, CD19, and CD20, followed by rabbit complement. Clonogenic assays were performed before and after purging. DNA was extracted and t(14;18) sequences amplified by PCR. PBMNCs collected by apheresis for back-up purposes were similarly evaluated. RESULTS Fifteen patients (25%) were PCR-positive before BM purging. Following MAb- and complement-mediated purging, there was a reduction in the PCR-amplified signal in 10 patients (67%). There was no reduction in colony-forming unit granulocyte-macrophage (CFU-GM) colony growth following purging. Eight of these 15 patients (53%) had morphologic evidence of BM involvement at the time of harvesting. In these eight patients, only three had a reduction in the PCR-amplified products, as compared with all seven who were morphologically negative at the time of BM harvesting (P = .026). Fourteen of these 15 patients had PBMNCs collected near the time of BM harvesting and 12 (86%) were PCR-positive. CONCLUSION BM purging with MAbs and complement results in reduction in the number of t(14;18)-positive tumor cells, especially in those patients who have no morphologic evidence of BM disease at the time of harvesting. Purged BM was less contaminated with t(14;18)-positive cells than unpurged PBMNCs, which were frequently contaminated with tumor cells.

No borna disease virus-specific RNA detected in blood of race horses and jockeys

2006 ◽  
Vol 18 (3-4) ◽  
pp. 177-180 ◽  
Author(s):  
Yong-Ku Kim ◽  
Kyung-Bo Noh ◽  
Chang-Su Han ◽  
Ju-Young Moon ◽  
Do-Kyung Yoon ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Disease Virus ◽  
Mononuclear Cells ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Peripheral Blood Mononuclear ◽  
Borna Disease Virus ◽  
Polymerase Chain ◽  
Race Horses ◽  
Borna Disease

Background:Borna disease virus (BDV) predominantly infects horses and sheep, causing a broad range of behavioural disorders. It is controversial whether BDV infects humans and causes psychiatric disorders.Objectives:We searched for BDV-derived nucleic acids in blood of race horses and jockeys riding the horses.Methods:We assayed for the BDV genome in RNA extracted from peripheral blood mononuclear cells (PBMC) of 39 race horses and 48 jockeys. Two polymerase chain reaction protocols [one-tube reverse transcription–polymerase chain reaction (RT-PCR) and two-step RT-PCR] were used to assay BDV p24 and p40 transcripts.Results:The p24 and p40 viral nucleic acid sequences were not detected in the PBMC RNAs from any of the race horses or jockeys.Conclusions:These data do not support an epidemiological association between BDV infection, race horses and humans.

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