Development and standardization of a new method for Virus-Like Particle quantification by digital PCR

Abstract BACKGROUND The analytically sensitive detection of KIT D816V in blood and bone marrow is important for diagnosing systemic mastocytosis (SM). Additionally, precise quantification of the KIT D816V variant allele fraction (VAF) is relevant clinically because it helps to predict multilineage involvement and prognosis in cases of advanced SM. Digital PCR (dPCR) is a promising new method for sensitive detection and accurate quantification of somatic mutations. METHODS We performed a validation study of dPCR for KIT D816V on 302 peripheral blood and bone marrow samples from 156 patients with mastocytosis for comparison with melting curve analysis after peptide nucleic acid-mediated PCR clamping (clamp-PCR) and allele-specific quantitative real-time PCR (qPCR). RESULTS dPCR showed a limit of detection of 0.01% VAF with a mean CV of 8.5% and identified the mutation in 90% of patients compared with 70% for clamp-PCR (P < 0.001). Moreover, dPCR for KIT D816V was highly concordant with qPCR without systematic deviation of results, and confirmed the clinical value of KIT D816V VAF measurements. Thus, patients with advanced SM showed a significantly higher KIT D816V VAF (median, 2.43%) compared with patients with indolent SM (median, 0.14%; P < 0.001). Moreover, dPCR confirmed the prognostic significance of a high KIT D816V VAF regarding survival (P < 0.001). CONCLUSIONS dPCR for KIT D816V provides a high degree of precision and sensitivity combined with the potential for interlaboratory standardization, which is crucial for the implementation of KIT D816V allele burden measurement. Thus, dPCR is suitable as a new method for KIT D816V testing in patients with mastocytosis.

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Immune Electron Microscopy (IEM) of a Canine Adeno-Associated Virus

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100051293 ◽

1974 ◽

Vol 32 ◽

pp. 256-257

Author(s):

Gunter F. Thomas ◽

M. David Hoggan

Keyword(s):

Electron Microscopy ◽

Cell Cultures ◽

Complement Fixation ◽

Hepatitis Virus ◽

Wide Distribution ◽

Immune Electron Microscopy ◽

Adeno Associated Virus ◽

Virus Like Particle ◽

Dog Kidney ◽

Green Monkeys

In 1968, Sugimura and Yanagawa described a small 25 nm virus like particle in association with the Matsuda strain of infectious canine hepatitis virus (ICHV). Domoto and Yanagawa showed that this particle was dependent on ICHV for its replication in primary dog kidney cell cultures (PDK) and was resistant to heating at 70°C for 10 min, and concluded that it was a canine adeno-associated virus (CAAV). Later studies by Onuma and Yanagawa compared CAAV with the known human serotypes (AAV 1, 2, 3) and AAV-4, known to be associated with African Green Monkeys. Using the complement fixation (CF) test, they found that CAAV was serologically related to AAV-3 and had wide distribution in the dog population of Japan.

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Selective Sampling and Orientation of Non-Homogeneous Tissues

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100100238 ◽

1968 ◽

Vol 26 ◽

pp. 98-99

Author(s):

C. C. Clawson ◽

L. W. Anderson ◽

R. A. Good

Keyword(s):

Electron Microscopy ◽

Electron Microscope ◽

Light Microscopy ◽

Random Sampling ◽

New Method ◽

Microscope Examination ◽

Small Areas ◽

Selective Sampling ◽

Large Numbers ◽

Specific Orientation

Investigations which require electron microscope examination of a few specific areas of non-homogeneous tissues make random sampling of small blocks an inefficient and unrewarding procedure. Therefore, several investigators have devised methods which allow obtaining sample blocks for electron microscopy from region of tissue previously identified by light microscopy of present here techniques which make possible: 1) sampling tissue for electron microscopy from selected areas previously identified by light microscopy of relatively large pieces of tissue; 2) dehydration and embedding large numbers of individually identified blocks while keeping each one separate; 3) a new method of maintaining specific orientation of blocks during embedding; 4) special light microscopic staining or fluorescent procedures and electron microscopy on immediately adjacent small areas of tissue.

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Hepatitis A Antigen in Liver, Bile and Stool

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100115328 ◽

1975 ◽

Vol 33 ◽

pp. 340-341

Author(s):

D.R. Jackson ◽

J.H. Hoofnagle ◽

A.N. Schulman ◽

J.L. Dienstag ◽

R.H. Purcell ◽

...

Keyword(s):

Hepatitis A ◽

Liver Enzymes ◽

Hepatitis A Virus ◽

Type A ◽

Initial Study ◽

Virus Like Particle ◽

Elevated Liver Enzymes ◽

Ag Particles ◽

Ag Particle ◽

Human Stool

Using immune electron microscopy Feinstone et. al. demonstrated the presence of a 27 nm virus-like particle in acute-phase stools of patients with viral hepatitis, type A, These hepatitis A antigen (HA Ag) particles were aggregated by convalescent serum from patients with type A hepatitis but not by pre-infection serum. Subsequently Dienstag et. al. and Maynard et. al. produced acute hepatitis in chimpanzees by inoculation with human stool containing HA Ag. During the early acute disease, virus like particles antigenically, morphologically and biophysically identical to the human HA Ag particle were found in chimpanzee stool. Recently Hilleman et. al. have described similar particles in liver and serum of marmosets infected with hepatitis A virus (HAV). We have investigated liver, bile and stool from chimpanzees and marmosets experimentally infected with HAV. In an initial study, a chimpanzee (no.785) inoculated with HA Ag-containing stool developed elevated liver enzymes 21 days after exposure.

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