ImageStream promyelocytic leukemia protein immunolocalization: In search of promyelocytic leukemia cells

Context and Aims:Rapid, accurate peripheral blood differentials are essential to maintain standards of patient care. CellaVision DM96 (CellaVision AB, Lund, Sweden) (CV) is an automated digital morphology and informatics system used to locate, pre-classify, store and transmit images of platelets, red and white blood cells to a trained technologist who confirms or edits CV cell classification. We assessed our experience with CV by evaluating sensitivity, specificity, positive predictive value and negative predictive value for CV in three different patient populations.Materials and Methods:We analyzed classification accuracy of CV for white blood cells, erythroblasts, platelets and artefacts over six months for three different university hospitals using CV.Results:CV classified 211,218 events for the adult cancer center; 51,699 events for the adult general hospital; and 8,009 events for the children's hospital with accuracy of CV being 93%, 87.3% and 95.4% respectively. Sensitivity and positive predictive value were <80% for immature granulocytes (band neutrophil, promyelocyte, myelocyte and metamyelocytes) (differences usually within one stage of maturation). Cell types comprising a lower frequency of the total events, including blasts, showed lower accuracy at some sites.Conclusions:The reduced immature granulocyte classification accuracy may be due in part to the subjectivity in classification of these cells, length of experience with the system and individual expertise of the technologist. Cells with low sensitivity and positive predictive value comprised a minority of the cells and should not significantly affect the technologist re-classification time. CV serves as a clinically useful instrument in performance of peripheral blood differentials.

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“…[1112] However, algorithms of sequential analysis with CV followed by other analytic techniques have yet to be described.…”

Section: Discussionmentioning

confidence: 99%

Experience with CellaVision DM96 for peripheral blood differentials in a large multi-center academic hospital system

Rollins‐Raval

Raval

Contis

2012

Journal of Pathology Informatics

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“…Recently we reported success using imaging flow cytometry to assess the intracellular localization and pattern of disrupted PML bodies in AML (11), which was subsequently confirmed by another research group to be a reliable technique (12). We therefore explored the applicability of this technology for discriminating cytoplasmic from nuclear NPM in AML.…”

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confidence: 88%

Detection of cytoplasmic nucleophosmin expression by imaging flow cytometry

et al. 2012

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Mutations within the nucleophosmin NPM1 gene occur in approximately one-third of cases of acute myeloid leukemia (AML). These mutations result in cytoplasmic accumulation of the mutant NPM protein. NPM1 mutations are currently detected by molecular methods. Using samples from 37 AML patients, we investigated whether imaging flow cytometry could be a viable alternative to this current technique. Bone marrow/peripheral blood cells were stained with anti-NPM antibody and DRAQ5 nuclear stain, and data were acquired on an ImageStream imaging flow cytometer (Amnis Corp., Seattle, USA). Using the similarity feature for data analysis, we demonstrated that this technique could successfully identify cases of AML with a NPM1 mutation based on cytoplasmic NPM protein staining (at similarity threshold of 1.1 sensitivity 88% and specificity 90%). Combining data of mean fluorescence intensity and % dissimilar staining in a 0-2 scoring system further improved the sensitivity (100%). Imaging flow cytometry has the potential to be included as part of a standard flow cytometry antibody panel to identify potential NPM1 mutations as part of diagnosis and minimal residual disease monitoring. Imaging flow cytometry is an exciting technology that has many possible applications in the diagnosis of hematological malignancies, including the potential to integrate modalities. ' 2012 International Society for Advancement of Cytometry Key termsimaging flow cytometry; acute leukemia; nucleophosmin; hematology NUCLEOPHOSMIN (NPM) is both a nuclear (predominantly nucleolar) and cytoplasmic protein that functions as a molecular chaperone (1). It has many functions including the prevention of protein aggregation in the nucleolus and regulation of p53 levels (2). Mutations within the C-terminal region (exon 12) of the NPM1 gene occur in approximately one-third of cases of de novo acute myeloid leukemia (AML) (3,4) leading to increased nuclear export and aberrant cytoplasmic accumulation of the mutant NPM protein (5). A number of methods have been assessed to detect cytoplasmic NPM (cNPM) in clinical specimens. Immunohistochemistry has been reported to be reliable, especially on B5-fixed bone marrow trephines, with close correlation between cNPM and the presence of mutated NPM1 (6). Immunocytochemistry of air-dried blood and bone marrow smears can also detect cNPM in myeloblasts but is insufficiently reliable for diagnostic use (7). Fluorescent confocal microscopy and standard flow cytometry have both been evaluated for cNPM detection (8,9). Standard flow cytometry cannot be used to assess the localization of molecules within specific cellular compartments, but two alternative approaches have been assessed. The first, using antibodies to mutant NPM1, was able to identify NPM1-mutated AML cases exclusively with or without the addition of a de novo NPM ''control'' antibody (8,9). The second method specifically addressed mean fluorescence intensity (MFI) and showed higher MFI in NPM1 mutated than wild-type AML cases (10). However, discrepancie...

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“…and laser-scanning cytometry (LSC; [12][13][14][15][16][17][18][19][20][21][22][23]. Developments based on time-delay integration, extended field-of-view for in focus imaging of the entire cell, and a wide range of detectable fluorophores, made ISC an excellent tool for statistically relevant imaging at a good resolution (21,22,(24)(25)(26)(27)(28)(29)(30). However, the in-flow approach can represent an obstacle for some applications (31,32) since cells are detached from their substrate, with the consequent loss of information about intercellular communication and/or interactions with the surrounding environment.…”

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confidence: 99%

A computational platform for robotized fluorescence microscopy (I): High‐content image‐based cell‐cycle analysis

Furia¹,

Pelicci²,

Faretta³

2013

Cytometry Pt A

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Hardware automation and software development have allowed a dramatic increase of throughput in both acquisition and analysis of images by associating an optimized statistical significance with fluorescence microscopy. Despite the numerous common points between fluorescence microscopy and flow cytometry (FCM), the enormous amount of applications developed for the latter have found relatively low space among the modern high-resolution imaging techniques. With the aim to fill this gap, we developed a novel computational platform named A.M.I.CO. (Automated Microscopy for Image-Cytometry) for the quantitative analysis of images from widefield and confocal robotized microscopes. Thanks to the setting up of both staining protocols and analysis procedures, we were able to recapitulate many FCM assays. In particular, we focused on the measurement of DNA content and the reconstruction of cell-cycle profiles with optimal parameters. Standard automated microscopes were employed at the highest optical resolution (200 nm), and white-light sources made it possible to perform an efficient multiparameter analysis. DNA-and protein-content measurements were complemented with image-derived information on their intracellular spatial distribution. Notably, the developed tools create a direct link between image-analysis and acquisition. It is therefore possible to isolate target populations according to a definite quantitative profile, and to relocate physically them for diffraction-limited data acquisition. Thanks to its flexibility and analysis-driven acquisition, A.M.I.CO. can integrate flow, image-stream and laser-scanning cytometry analysis, providing high-resolution intracellular analysis with a previously unreached statistical relevance. ' 2013 International Society for Advancement of Cytometry Key termsimage-cytometry; cell-cycle; automated microscopy; image analysis LIMITED sample availability, as for material of clinical origin, and a poor statistical representation of targeted cell populations, for example, stem cells, can make the comprehension of biological heterogeneity a very challenging task (1-4). High-content approaches have been consequently developed to extend the number of simultaneously analyzable parameters (5-7).Flow cytometry (FCM) is a powerful technique that provides statistically relevant measurements. Quantification at single-cell level, elevated content, fast acquisition, and contained analysis time make it a very valuable tool for the identification of rare cell populations. In addition, enormous advances in limiting the required amount of sample per analysis have also been achieved by system miniaturization (7-11).This excellent "statistical resolution" is, however, coupled to a complete absence of an intracellular view and, to date, fluorescence microscopy maintains its leadership in providing a high-resolution description of the inner cell compartments. Nevertheless, technological efforts have tried to fill the imaging gap between FCM and fluorescence microscopy, leading to the development of ...

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ImageStream promyelocytic leukemia protein immunolocalization: In search of promyelocytic leukemia cells

Cited by 13 publications

References 14 publications

Experience with CellaVision DM96 for peripheral blood differentials in a large multi-center academic hospital system

Experience with CellaVision DM96 for peripheral blood differentials in a large multi-center academic hospital system

Detection of cytoplasmic nucleophosmin expression by imaging flow cytometry

A computational platform for robotized fluorescence microscopy (I): High‐content image‐based cell‐cycle analysis

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