Microfluidics enables multiplex evaluation of the same cells for further studies.

OBJECTIVE: The continuous discovery of biomarkers and their evolving use for the diagnosis and guidance of therapy for patients with cancer has increased awareness of the need to triage biospecimens properly. On occasion, cytology samples are the only type of biospecimen available for analysis. Often, the current approach for these latter specimens is cytopathology-centric, with cells limited to examination by bright field microscopy. When specimens are paucicellular, there is often insufficient material for ancillary testing. Therefore, a need exists to develop an alternative approach that allows for the multiplexed analysis of cells when they are limited in number. In recent previous publications, we demonstrated that clinically derived cells from tissue are suitable for evaluation in a microfluidic device. In our current endeavour, we seek to expand upon those findings and determine if those same cells can be recovered for further analysis. METHODS: A microfluidic channel was designed, fabricated and tested using cytology specimens generated from tissue specimens. The cytological features of the cells tested were examined prior to entering the channel; they were then compared to similar cells while in the channel, and upon recovery from the channel. Recovery of DNA and proteins were also tested. RESULTS: The morphology of the tested cells was not compromised in either the channel or upon recovery. More importantly, the integrity of the cells remained intact, with the recovery of proteins and high molecular weight DNA possible. CONCLUSIONS: We developed and tested an alternative approach to the processing of cytopathology specimens that enables multiplexed evaluation. Using microfluidics, cytological examination of biopecimens can be performed, but in contrast to existing approaches, the same cells examined can be recovered for downstream analysis.

Comparable cytological features between cells processed as either wet mount or conventional cytospins.

OBJECTIVE: Microfluidics represents a novel approach for the processing of pathological biopsy specimens. It represents an enabling platform between traditional morphology-driven pathology diagnostics and future sequencing technologies. Microfluidics requires the dissociation of cells from tissue, but the evaluation of these cells still lies within the domain of pathology, specifically cytopathology. The dissociated cells, however, require supravital staining and examination under wet mount conditions as part of their processing in a microfluidic platform. These conditions are vastly different from current approaches for cytopathological specimen preparation. No studies to date have compared the cytological characteristics of cells between these two different conditions. METHODS: Slivers of different types of unfixed tissue were procured and the cells were dissociated. The cells were recovered and separated for processing either by staining with a supravital dye and examination under wet mount conditions or by conventional preparatory methods and staining by the Papanicolaou method. RESULTS: Obvious differences existed in the tinctorial hue between similar cells stained by the two different methods; however, there were no significant differences in the features between the matched cells. Some cells in the wet mount preparations existed in three-dimensional balls, reducing the quality of the images relative to cells flattened to the slide by the cytospin method. CONCLUSIONS: Cells stained with a supravital stain and examined in a wet mount environment appear to be cytologically similar to cells prepared by conventional methods. Long-established criteria for the evaluation of cells prepared by established protocols can therefore be extended and applied to cells viewed within a microfluidic platform.

An evaluation of synthetic and natural supravital stains for the cytological examination of dissociated cells in a microfluidic channel.

OBJECTIVE: Advances in biotechnology will result in paradigm shifts in both oncology and diagnostics. In pathology, methods such as microfluidics are being explored as delivery tools so that processed cells can serve dual purposes: conventional cytology-based diagnostics and recovery of the same cells for molecular assays. This wet mount-based approach to diagnosis will require staining of these cells by supravital dyes. This study was undertaken to determine the optimal supravital stain for the examination of cells in the wet mount preparations present in microfluidic platforms. METHODS: Cells were dissociated from portions of tissue similar in size to a traditional core biopsy. These tissue-free cells were separately examined with two synthetic dyes and two natural dyes at varying dilutions. RESULTS: Different dilutions of the synthetic dyes toluidine blue and methylene blue resulted in varying degrees of staining, whereas different dilutions of the natural dyes resulted in fairly constant intensities of colour. These characteristics affected the visualization of cells in wet mount preparations: optimally titered synthetic dyes gave better nuclear detail and cytoplasmic contrast. CONCLUSIONS: All four dyes stained the test cells, but to different degrees and intensities. In our assessment, optimally titered synthetic dyes were better suited to the wet mount approach of microfluidics when compared with natural dyes.

CD117+ small cell lung cancer lacks the asp 816-->val point mutation in exon 17.

AIMS: To determine the frequency of point mutation in c-kit in CD117+ small cell lung cancer (SCLC). A significant proportion of SCLCs have been documented to be CD117+, thereby signifying they express the c-kit gene product. This finding suggests this tumour may be a potential target for tyrosine kinase inhibitor (TKI) agents directed at c-kit. A point mutation in exon 17 of the c-kit gene, however, can abrogate the binding of TKIs. This being the case, immunohistochemistry is necessary to identify potential candidates for treatment with TKIs, but DNA sequence analysis may need to be performed to determine if these tumours will respond. METHODS AND RESULTS: Tumour cells of 23 cases of SCLC showing immunoreactivity for CD117 were laser capture microdissected from archived formalin-fixed paraffin-embedded tissue and the DNA isolated. PCR on exon 17 of the c-kit gene was performed and the amplified product sequenced. No point mutations were detected. CONCLUSIONS: The absence of mutations in exon 17 of CD117+ SCLC suggests this tumour may respond to therapy with TKI.

Elastofibroma dorsi: elaboration of cytologic features and review of its pathogenesis.

Elastofibroma is a slow-growing soft tissue lesion characteristically found between the inferior scapula and chest wall. Because it behaves clinically in a benign manner, fine-needle aspiration (FNA) represents the simplest and quickest method of obtaining a definitive diagnosis, thus obviating more invasive means of obtaining a tissue diagnosis. However, due to the nature of this lesion a correct diagnosis can inadvertently be missed. Herein we describe the findings of a recent FNA that obtained abundant diagnostic material and elaborate upon the spectrum of cytologic features of the elastic fibers that can be identified. These features should be recognized, since aspiration biopsy in elastofibromas can lead to hypocellular smears. In addition, we discuss recent developments in the pathophysiology of elastic fibers and their application toward understanding the generation of an elastofibroma.

Epithelioid angiomyolipoma: appearance on fine-needle aspiration report of a case.

Epithelioid angiomyolipoma is a recently recognized clinicopathologic entity first described by Martignoni et al. in 1995. Since then, several articles have further clarified its histogenesis and histologic features. Due to the presence of polygonal cells with voluminous cytoplasms, this neoplasm is often mistaken for renal-cell carcinoma. In this case presentation, we describe the cytologic features of an epithelioid angiomyolipoma obtained by fine-needle aspiration. The histogenesis and how it relates to diagnosis is briefly discussed. The importance of ancillary techniques in the differential diagnosis of epithelioid cells obtained in a renal aspirate is reviewed.