Feasibility of single-cell analysis of model cancer and foetal cells in blood after isolation by cell picking.

The objective of the present feasibility study was to transfer single cell line cells to either microscopy slides for downstream immune characterization or to polymerase chain reaction tubes for downstream DNA quantitation. Tumour cell lines, SKBR3 and MCF7 and trophoblast cell line JEG-3 were spiked in healthy donor blood. The CytoTrack system was used to scan the spiked blood samples to identify target cells. Individual target cells were identified, picked by use of a CytoPicker and deposited to either a microscopic slide or a polymerase chain reaction tube (PCR). Single tumour cells on microscopic slides were further immunostained with human epidermal growth factor receptor 2 (Her2) and epithelial cell adhesion molecule (EpCAM). From the picked cells in polymerase chain reaction tubes, DNA was amplified, quantified and used for Short Tandem Repeat genotyping. Depositing rare cells to microscopy slides was laborious with only five cells per hour. In this study with a trained operator, the picked cells had an 80.5% recovery rate. Depositing single trophoblast cells in PCR tubes was a faster process with 10 cells in 5 min. Immunostaining of isolated cells by both Her2 and EpCAM was possible but showed varying staining intensity. Presence of trophoblasts and contaminating white blood cells in PCR tubes after cell picking was confirmed based on DNA yield and mixed Short Tandem Repeat profiles in five out of eight samples. Using the CytoPicker tool, single tumour and trophoblast cells were successfully isolated and moved from blood samples, allowing subsequent immunostaining or Short Tandem Repeat genotyping.

Fluorescence in situ hybridization (FISH) in the microbiological diagnostic routine laboratory: a review.

Early identification of microbial pathogens is essential for rational and conservative antibiotic use especially in the case of known regional resistance patterns. Here, we describe fluorescence in situ hybridization (FISH) as one of the rapid methods for easy identification of microbial pathogens, and its advantages and disadvantages for the diagnosis of pathogens in human infections in the laboratory diagnostic routine. Binding of short fluorescence-labeled DNA or nucleic acid-mimicking PNA probes to ribosomes of infectious agents with consecutive analysis by fluorescence microscopy allows identification of bacterial and eukaryotic pathogens at genus or species level. FISH analysis leads to immediate differentiation of infectious agents without delay due to the need for microbial culture. As a microscopic technique, FISH has the unique potential to provide information about spatial resolution, morphology and identification of key pathogens in mixed species samples. On-going automation and commercialization of the FISH procedure has led to significant shortening of the time-to-result and increased test reliability. FISH is a useful tool for the rapid initial identification of microbial pathogens, even from primary materials. Among the rapidly developing alternative techniques, FISH serves as a bridging technology between microscopy, microbial culture, biochemical identification and molecular diagnostic procedures.

Cryopreservation of Circulating Tumor Cells for Enumeration and Characterization.

BACKGROUND: A blood sample containing circulating tumor cells (CTCs) may serve as a surrogate for metastasis in invasive cancer. Cryopreservation will provide new opportunities in management of clinical samples in the laboratory and allow collection of samples over time for future analysis of existing and upcoming cancer biomarkers. METHODS: Blood samples from healthy volunteers were spiked with high (∼500) and low (∼50) number of tumor cells from culture. The samples were stored at -80C with cryopreservative dimethyl sulfoxide mixed with Roswell Park Memorial Institute 1640 medium. Flow cytometry tested if cryopreservation affected specific biomarkers regularly used to detect CTCs, i.e. cytokeratin (CK) and epithelial cell adhesion molecule (EpCAM) and white blood cell specific lymphocyte common antigen (CD45). After various time intervals (up to 6 months), samples were thawed and tumor cell recovery (enumeration) was examined. Clinical samples may differ from cell line studies, so the cryopreservation protocol was tested on 17 patients with invasive breast cancer and tumor cell recovery was examined. Two blood samples were drawn from each patient. RESULTS: Biomarkers, CK, CD45, and EpCAM, were not affected by the freezing and thawing procedures. Cryopreserved samples (n = 2) spiked with a high number of tumor cells (∼500) had a ∼90% recovery compared with the spiked fresh samples. In samples spiked with lower numbers of tumor cells (median = 43 in n = 5 samples), the recovery was 63% after cryopreservation (median 27 tumor cells), p = 0.03. With an even lower number of spiked tumor cells (median = 3 in n = 8 samples), the recovery rate of tumor cells after cryopreservation did not seem to be affected (median = 8), p = 0.09. Time of cryopreservation did not affect recovery. When testing the effect of cryopreservation on enumeration in clinical samples, no difference was observed in the number of CTCs between the fresh and the cryopreserved samples based on n = 17 pairs, p = 0.83; however, the variation was large. This large variation was confirmed by clinically paired fresh samples (n = 64 pairs), where 95% of the samples (<30 CTCs) vary in number up to ±15 CTCs, p = 0.18. CONCLUSIONS: A small loss of CTCs after cryopreservation may be expected; however, cryopreservation of CTCs for biomarker characterization for clinical applications seems promising.

Retracing Circulating Tumour Cells for Biomarker Characterization after Enumeration.

BACKGROUND: Retracing and biomarker characterization of individual circulating tumour cells (CTCs) may potentially contribute to personalized metastatic cancer therapy. This is relevant when a biopsy of the metastasis is complicated or impossible to acquire. METHODS: A novel disc format was used to map and retrace individual CTCs from breast-cancer patients and nucleated cells from healthy blood donors using the CytoTrack platform. For proof of the retracing concept, CTC HER2 characterization by immunofluorescence was tested. RESULTS: CTCs were detected and enumerated in three of four blood samples from breast-cancer patients and the locations of each individual CTCs were mapped on the discs. Nucleated cells were retraced on seven discs with 96.6%±8.5% recovery on five fields of view on each disc. Shifting of field of view for retracing was measured to 4-29 µm. In a blood sample from a HER2-positive breast-cancer patient, CTC enumeration and mapping was followed by HER2 characterization and retracing to demonstrate downstream immunofluorescence analysis of the CTC. CONCLUSION: Mapping and retracing of CTCs enables downstream analysis of individual CTCs for existing and future cancer genotypic and phenotypic biomarkers. Future studies will uncover this potential of the novel retracing technology.

[Identification of Mycobacterium species from BACTEC MGIT™ positive cultures with Oligo-FISH and PNA-FISH methods]. / BACTEC MGIT™ Pozitif Kültürlerden Mycobacterium türlerinin Oligo-FISH ve PNA-FISH Yöntemleriyle Tanimlanmasi.

Rapid and accurate diagnosis of mycobacteria is very important in the prevention and effective treatment of tuberculosis which is still a serious public health problem. Fluorescence in situ hybridization (FISH) method using rRNA targeted probes allows for precise and accurate identification of mixed microorganisms from cultures and directly from clinical samples within a few hours without the need for culture methods. In this study it was aimed to compare the diagnostic performance of two different FISH methods (Oligo-FISH and PNA-FISH) with the conventional culture methods for the identification of Mycobacterium spp. grown in BACTEC MGIT™ (Mycobacteria Growth Indicator Tube) system. A total of 60 MGIT (BD, USA) positive, 52 MGIT negative samples and 10 different reference strains were included in the study. 16S rRNA targeted oligonucleotide probes (Myc657: Mycobacterium subdivision, Eub338: Positive control, NonEub: Negative control) were used for oligo-FISH, and 16S rRNA targeted peptide nucleotide probes (MTC: Mycobacterium tuberculosis complex, NTM: Non-tuberculosis Mycobacterium, BacUni: Positive control) for PNA-FISH. Ehrlich-Ziehl-Neelsen staining (ARB) and Löwenstein-Jensen (LJ) culture methods were performed as conventional methods as well as MGIT 960 culture system. Of MGIT positive 60 samples (44 sputum, 4 tissue, 4 urine, 3 bronchoalveolar lavage, 3 CSF, 1 abscess, 1 peritoneal fluid), 29 (48.3%) were found positive for ARB and 44 (73.3%) with LJ culture methods giving a total of 59 positive results. Fifty-eight (96.6%) of those isolates were identified as MTC, and one (1.7%) as NTM by conventional methods. By using Oligo-FISH, 95% (57/60) of the isolates were identified as Mycobacterium spp., while three samples (5%) yielded negative result. By using PNA-FISH, 54 (91.5%) isolates were identified as mycobacteria, of them 53 (90%) were typed as MTC and 1 (1.7%) as NTM. Five isolates that were found positive with Oligo-FISH, but negative with PNA-FISH, yielded positive result with PNA-FISH method performed with minor modifications. It was determined that both FISH methods are more rapid (approximately 2-2.5 hours) and practical than the conventional culture methods and also PNA-FISH was more practical than Oligo-FISH. The sensitivity, specificity, positive and negative predictive values of the probes used for Oligo-FISH, were 96.6%, 100%, 100% and 96.4%, respectively. Those values for the probes used for PNA-FISH, were 91.5%, 100%, 100% and 91.4%, respectively (p< 0.0001). The compatibility of the methods was calculated with kappa statistical analysis, assigning perfect concordances between Oligo- and PNA-FISH methods, as well as between conventional and both of the FISH methods (κ: 0.964, 0.929, 0.964; p= 0.001). The coverage of oligonucleotide and PNA probes was also checked by using 16S rRNA gene sequence database retrieved from the SILVA 102. It was determined that the rates of coverage were 86.5% for Eub338, 41.7% for Myc657, 84.2% for BacUni, 76.3% for MTC (100% for only M.tuberculosis and M.bovis) and 25.8% for NTM probes. In conclusion, Oligo- and PNA-FISH methods seem to be successful for rapid and accurate identification of Mycobacterium spp. from MGIT positive cultures in routine mycobacteriology laboratories without the need for expensive methods.

PNA fluorescent in situ hybridization (FISH) for rapid microbiology and cytogenetic analysis.

Hybridization-based assays for the detection of nucleic acids including in situ hybridization are increasingly being utilized in a wide variety of disciplines such as cytogenetics, microbiology, and histology. Generally in situ hybridization assays utilize either cloned genomic probes for the detection of DNA sequences or oligonucleotide probes for the detection of DNA or RNA sequences. Alternately, PNA probes are increasingly being utilized in a variety of in situ hybridization assays. The neutral backbone of the PNA molecule allows for the PNA probes to bind to DNA or RNA under low ionic strength conditions that will either disfavor reannealing of complimentary genomic sequences or are denaturing for RNA secondary structure but are favorable for PNA/DNA or PNA/RNA hybridization. For in situ hybridization assays these unique properties of PNA probes offer significant advantages that allow for the development of fast, simple, and robust assays (Figs. 14.1 and 14.2).

Evaluation of mupA EVIGENE assay for determination of high-level mupirocin resistance in Staphylococcus aureus.

Mupirocin is widely used to decolonize patients carrying Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA). The aim of this study was to determine the presence of high-level mupirocin resistance by a new commercially available mupA genotypic diagnostic product, mupA EVIGENE assay (AdvanDx).

Rapid and accurate identification of Candida albicans isolates by use of PNA FISHFlow.

We developed the simple, rapid (1 h), and accurate PNA FISH(Flow) method for the identification of Candida albicans. The method exploits unique in solution in situ hybridization conditions under which the cells are simultaneously fixed and hybridized. This method facilitates the accurate identification of clinical yeast isolates using two scoring techniques: flow cytometry and fluorescence microscopy.

Multicenter evaluation of the Candida albicans/Candida glabrata peptide nucleic acid fluorescent in situ hybridization method for simultaneous dual-color identification of C. albicans and C. glabrata directly from blood culture bottles.

We evaluated the performance of the Candida albicans/Candida glabrata peptide nucleic acid fluorescent in situ hybridization (PNA FISH) method, a rapid two-color assay for detection of C. albicans and C. glabrata, in a multicenter study. The assay is designed for use directly from positive blood culture bottles in a FISH format. Intact, fixed cells are labeled fluorescent green (C. albicans) or fluorescent red (C. glabrata) by rRNA hybridization of fluorophore-labeled PNA probes. Results are available <3 h after cultures signal positive. An evaluation of 197 routine blood culture bottles newly positive for yeast by Gram staining was performed at five hospitals. The sensitivities of detection for C. albicans, and C. glabrata were 98.7% (78/79) and 100% (37/37), respectively, and the specificity for both components of the assay was 100% (82/82). The assay was also evaluated with 70 fungal reference strains and was challenged in the BacT/ALERT microbiological detection system with spiked blood culture bottles. These results support the use of the assay for rapid, simultaneous identification of C. albicans and C. glabrata in positive blood culture bottles. This rapid assay may aid in the selection of initial antifungal drugs, leading to improved patient outcomes.

Peptide nucleic acid fluorescence in situ hybridization for rapid detection of Klebsiella pneumoniae from positive blood cultures.

This study evaluated a novel peptide nucleic acid (PNA) probe targeting a region of the 23S rRNA gene of Klebsiella pneumoniae by fluorescence in situ hybridization (FISH). Analytical performance was determined using 39 reference strains and other well-characterized strains of Klebsiella spp. and Enterobacter aerogenes. The probe was found to be specific for the K. pneumoniae complex (K. pneumoniae including Klebsiella ozaenae and Klebsiella variicola). The diagnostic accuracy was evaluated with 264 blood cultures containing Gram-negative rods. Using conventional identification as the reference, performance specifications were as follows: sensitivity 98.8 %, specificity 99.5 %, positive predictive value 98.8 % and negative predictive value 99.5 %. Discrepancies were resolved by PNA FISH retest and phenotypic tests. In conclusion, the K. pneumoniae probe provided an accurate diagnosis within 3 h and may supplement other methods for direct identification of Gram-negative bacteria.

Rapid identification of Staphylococcus aureus in blood cultures by a combination of fluorescence in situ hybridization using peptide nucleic acid probes and flow cytometry.

Fluorescence in situ hybridization (FISH) using peptide nucleic acid probes (PNAs) allows the identification of Staphylococcus aureus from human blood culture samples. We present data revealing that the combination of PNA FISH and flow cytometry is a possible approach for the noncultural identification of staphylococci in blood cultures.

PNA FISH: an intelligent stain for rapid diagnosis of infectious diseases.

Fluorescence in situ hybridization using peptide nucleic acid probes (PNA FISH) is a novel diagnostic technique combining the simplicity of traditional staining procedures with the unique performance of PNA probes to provide rapid and accurate diagnosis of infectious diseases; a feature that makes PNA FISH well suited for routine application and enables clinical microbiology laboratories to report important information for patient therapy within a time frame not possible using classic biochemical methods. Having transitioned from an academic curiosity into an advanced diagnostic tool, PNA probes are now debuting on the infectious disease stage, representing the new generation of therapy-directing diagnostics.

Direct identification of Staphylococcus aureus from positive blood culture bottles.

Fluorescence in situ hybridization (FISH) using peptide nucleic acid (PNA) probes targeting Staphylococcus aureus 16S rRNA is a novel method for direct identification of S. aureus from positive blood culture bottles. The test (S. aureus PNA FISH) is performed on smears made directly from positive blood culture bottles with gram-positive cocci in clusters (GPCC) and provides results within 2.5 h. A blinded comparison of S. aureus PNA FISH with standard identification methods was performed in collaboration with eight clinical microbiology laboratories. A total of 564 routine blood culture bottles positive for GPCC recovered from both aerobic and anaerobic media from three different manufacturers (ESP, BACTEC, and BacT/Alert) were included in the study. The sensitivity and specificity of S. aureus PNA FISH were 100% (57 of 57) and 99.2% (116 of 117), respectively, with 174 GPCC-positive ESP blood culture bottles, 98.5% (67 of 68) and 98.5% (129 of 131), respectively, with 200 GPCC-positive BACTEC blood culture bottles, and 100% (74 of 74) and 99.1% (115 of 116), respectively, with 190 GPCC-positive BacT/Alert blood culture bottles. It is concluded that S. aureus PNA FISH performs well with commonly used continuously monitoring blood culture systems.

Fluorescence in situ hybridization with peptide nucleic acid probes for rapid identification of Candida albicans directly from blood culture bottles.

A new fluorescence in situ hybridization (FISH) method that uses peptide nucleic acid (PNA) probes for identification of Candida albicans directly from positive-blood-culture bottles in which yeast was observed by Gram staining (herein referred to as yeast-positive blood culture bottles) is described. The test (the C. albicans PNA FISH method) is based on a fluorescein-labeled PNA probe that targets C. albicans 26S rRNA. The PNA probe is added to smears made directly from the contents of the blood culture bottle and hybridized for 90 min at 55 degrees C. Unhybridized PNA probe is removed by washing of the mixture (30 min), and the smears are examined by fluorescence microscopy. The specificity of the method was confirmed with 23 reference strains representing phylogenetically related yeast species and 148 clinical isolates covering the clinically most significant yeast species, including C. albicans (n = 72), C. dubliniensis (n = 58), C. glabrata (n = 5), C. krusei (n = 2), C. parapsilosis (n = 4), and C. tropicalis (n = 3). The performance of the C. albicans PNA FISH method as a diagnostic test was evaluated with 33 routine and 25 simulated yeast-positive blood culture bottles and showed 100% sensitivity and 100% specificity. It is concluded that this 2.5-h method for the definitive identification of C. albicans directly from yeast-positive blood culture bottles provides important information for optimal antifungal therapy and patient management.

Rapid identification of Staphylococcus aureus directly from blood cultures by fluorescence in situ hybridization with peptide nucleic acid probes.

A new fluorescence in situ hybridization (FISH) method with peptide nucleic acid (PNA) probes for identification of Staphylococcus aureus directly from positive blood culture bottles that contain gram-positive cocci in clusters (GPCC) is described. The test (the S. aureus PNA FISH assay) is based on a fluorescein-labeled PNA probe that targets a species-specific sequence of the 16S rRNA of S. aureus. Evaluations with 17 reference strains and 48 clinical isolates, including methicillin-resistant and methicillin-susceptible S. aureus species, coagulase-negative Staphylococcus species, and other clinically relevant and phylogenetically related bacteria and yeast species, showed that the assay had 100% sensitivity and 96% specificity. Clinical trials with 87 blood cultures positive for GPCC correctly identified 36 of 37 (97%) of the S. aureus-positive cultures identified by standard microbiological methods. The positive and negative predictive values were 100 and 98%, respectively. It is concluded that this rapid method (2.5 h) for identification of S. aureus directly from blood culture bottles that contain GPCC offers important information for optimal antibiotic therapy.

PNA for rapid microbiology.

The acceptance of rRNA sequence diversity as a criterion for phylogenetic discrimination heralds the transition from microbiological identification methods based on phenotypic markers to assays employing molecular techniques. Robust amplification assays and sensitive direct detection methods are rapidly becoming the standard protocols of microbiology laboratories. The emergence of peptide nucleic acid (PNA) from its status as an academic curiosity to that of a promising and powerful molecular tool, coincides with, and complements, the transition to rapid molecular tests. The unique properties of PNA enable the development of assay formats, which go above and beyond the possibilities of DNA probes. PNA probes targeting specific rRNA sequences of yeast and bacteria with clinical, environmental, and industrial value have recently been developed and applied to a variety of rapid assay formats. Some simply incorporate the sensitivity and specificity of PNA probes into traditional methods, such as membrane filtration and microscopic analysis; others involve recent techniques such as real-time and end-point analysis of amplification reactions.