Wiebke Laffers

Iterative Restaining as a Pivotal Tool for n-Color Immunophenotyping by Slide-Based Cytometry

Slide‐based cytometry (SBC) allows to “ask a cell a second time.” We used this tool for detailed immunophenotyping of peripheral blood leukocytes (PBLs).

Hyperspectral imaging of mucosal surfaces in patients.

The aim of this study was to proof applicability of hyperspectral imaging for the analysis and classification of human mucosal surfaces in vivo. The larynx as a prototypical anatomically well-defined surgical test area was analyzed by microlaryngoscopy with a polychromatic lightsource and a synchronous triggered monochromatic CCD-camera. Image stacks (5 benign, 7 malignant tumors) were analyzed by established software (principal component analysis PCA, hyperspectral classification, spectral profiles). Hyperspectral image datacubes were analyzed and classified by conventional software. In PCA, images at 590-680 nm loaded most onto the first PC which typically contained 95% of the total information. Hyperspectral classification clustered the data highlighting altered mucosa. The spectral profiles clearly differed between the different groups. Hyperspectral imaging can be applied to mucosal surfaces. This approach opens the way to analyze spectral characteristics of histologically different lesions in order to build up a spectral library and to allow non-touch optical biopsy.

Clinical applications of slide-based cytometry--an update.

Slide-based cytometric approaches open the possibility to obtain quantitative and objective data from specimens that so far have not been accessible to this kind of analysis. In this review, we will highlight the specific advantages of slide-based cytometry (SBC) and show the applications that have been established for clinical samples. Focuses are cytomic analyses of oncological and hematological samples where the slide-based concept turned out to open new dimensions in understanding underlying cellular networks. We review the recent literature and point out future applications.

Concepts for Absolute Immunophenosubtyping by Slide- Based Cytometry

Differential immunophenotyping is essential for diagnosis and follow-up. Routine analysis requires a minimum of 250 γl blood, and neither a detailed immunophenosubtyping nor absolute cell count are available. We present two concepts using slide-based cytometry in order to break these limitations. Materials and Methods: 10 γl EDTA blood were stained with DRAQ5 and either CD3-PE, CD4-Alexa Fluor 488 and CD8-PE-Cy5 or CD45-FITC and CD14-PE. A ‘no-lyse-no-wash’ versus a ‘lyse-no-wash’ method (Quicklysis) was performed. 20 γl of the suspension were applied to a Neubauer chamber. Leukocytes were analyzed by laser scanning cytometry (LSC) twice with a time interval of 15 min. Another aliquot of blood was taken for routine analysis. Erythrocyte count was performed after another dilution. Results: Brévais’ regression coefficient showed a good internal correlation of LSC (r > 0.85) with no significant difference between routine analysis and LSC (‘lyse-no-wash’: r = 0.9, p = 0.009, α = 0.05; ‘no-lyse-no-wash’: r = 0.98, p < 0,0001, α = 0.05). Immunophenosubtyping by LSC (CD3/CD4/ CD8, CD45/CD14) was unequivocal. Conclusion: A detailed immunophenosubtyping and absolute cell count is possible by using the Neubauer chamber for slide-based analysis. This method requires minimal amounts of blood, is very cost-efficient, and data show no significant difference to routine laboratory. This concept might prove versatile in patients with low blood volume and where absolute cell count is essential.

Development of an image pre‐processor for operational hyperspectral laryngeal cancer detection

Hyperspectral imaging (HSI) is a technology with high potential in the field of non-invasive detection of cancer. However, in complex imaging situations like HSI of the larynx with a rigid endoscope, various image interferences can disable a proper classification of cancerous tissue. We identified three main problems: i) misregistration of single images in a HS cube due to patient heartbeat ii) image noise and iii) specular reflections (SR). Consequently, an image pre-processor is developed in the current paper to overcome these image interferences. It encompasses i) image registration ii) noise removal by minimum noise fraction (MNF) transformation and iii) a novel SR detection method. The results reveal that the pre-processor improves classification performance, while the newly developed SR detection method outperforms global thresholding technique hitherto used by 46%. The novel pre-processor will be used for future studies towards the development of an operational scheme for HS-based larynx cancer detection. RGB image of the larynx derived from the hyperspectral cube and corresponding specular reflections (a) manually segmented and (b) detected by a novel specular reflection detection method.

Six and more color immunophenotyping on the slide by laser scanning cytometry (LSC)

The request for a more profound immunophenotyping and sometimes the lack of material demands more measurable fluorescence colors to increase the number of detectable antigens per specimen. Six different fluorescences are distinguishable in the Laser Scanning Cytometer (LSC). In the present study we wanted to increase this number to eight colors per measurement. Combined with an earlier study it is likely possible to measure n fluorescences i.e. n leukocyte subsets by a series of measurements followed by subsequent restraining steps. The new method is realized by s-ing the combination of filter change and a subsequent re-measurement for the distinction between the fluorescent dyes Cy5 and Cy5.5. The optical filters are replaced after the first measurement and the same specimen is remeasured without removing it from the microscope. For the second measurement a filter is inserted that detects Cy5.5 but not Cy5 (710/10nm). After the second measurement of the same specimen both data files are combined. With the aid of this feature it is possible to line out the differences between both measurements. If the data from the second measuring (Cy5.5 only) is subtracted from the first, Cy5 data is the result. After the first two measurements when eight different fluorescences (i.e. antigens or leukocyte subsets) were analyzed, the same cells are restained and a new measurement is performed. In theory, one can perform n re-measurements with eight fluorescences respectively. The information gained per specimen is only limited by the number of available antibodies and b sterical hindrance.

Hyperspectral Imaging Using Flexible Endoscopy for Laryngeal Cancer Detection.

Hyperspectral imaging (HSI) is increasingly gaining acceptance in the medical field. Up until now, HSI has been used in conjunction with rigid endoscopy to detect cancer in vivo. The logical next step is to pair HSI with flexible endoscopy, since it improves access to hard-to-reach areas. While the flexible endoscope’s fiber optic cables provide the advantage of flexibility, they also introduce an interfering honeycomb-like pattern onto images. Due to the substantial impact this pattern has on locating cancerous tissue, it must be removed before the HS data can be further processed. Thereby, the loss of information is to minimize avoiding the suppression of small-area variations of pixel values. We have developed a system that uses flexible endoscopy to record HS cubes of the larynx and designed a special filtering technique to remove the honeycomb-like pattern with minimal loss of information. We have confirmed its feasibility by comparing it to conventional filtering techniques using an objective metric and by applying unsupervised and supervised classifications to raw and pre-processed HS cubes. Compared to conventional techniques, our method successfully removes the honeycomb-like pattern and considerably improves classification performance, while preserving image details.

Novel approaches for immunophenotyping by laser scanning cytometry (LSC)

LSC is a microscope-based technology. The principle of the instrument is that any specimen is immobilized on a microscope slide. Therefore the cells are not lost in a fluid stream but are kept on the slide and minimal specimens as low as 1.000 cells can be analyzed. Additionally cells are available for further analyses such as staining for another set of specific markers and re-analysis or cytological staining (H&E). This approach multiplies the information gained from a given sample. We have established an assay for immunophenotyping of peripheral blood leukocytes by LSC. Cells are prepared according to routine flow cytometry protocols with a first set of CD-antibodies and are fixed on microscope slides. As a stable trigger signal the nuclear DNA is stained by 7-aminoactinomycin-D. This guarantees that all nucleated cells and that only nucleated cells are included in the analysis, and many differentiate between lymphocytes and neutrophiles by staining intensity. After analysis cells are stained with a second set of CD-antibodies and analyzed again. This step can be repeated with a third set of CD-antigens. Since the location of the cells on the slide is fixed data from the analyses can be attributed to the same cell.

HISTOLOGIC DIAGNOSES IN PERSISTENTLY SWOLLEN CERVICAL LYMPH NODES

Biopsy and histological examination of persistently enlarged cervical lymph nodes represent a major health care issue and have high impact on further clinical therapy. Tertiary health centers are faced with an increased demand for diagnostic workup to rule out malignancy. We performed a retrospective study from January 2000 to June 2008 to identify patients referred to us for diagnostic biopsy and to document the histopathological result.

Früherkennung kanzeröser Läsionen in Oropharynx und Mundhöhle

BACKGROUND Early detection of cancerous lesions is still crucial for a patients prognosis. Although diagnostic access to the oral cavity and oropharynx is comparably easy, the incidence of resulting disease remains high. This is due to the fact that in many cases, malignity is recognized too late on a purely visual basis. Previously, we discussed the application of hyperspectral imaging for early detection of precancerous and cancerous lesions of the larynx. This time, we evaluate the method in the oral cavity and oropharynx. MATERIALS AND METHODS In 85 patients scheduled for endoscopy, hyperspectral imaging was performed. We used a rigid 0-degree endoscope, a light-adjustable monochromator, and a hyperspectral camera. For evaluation of the method, 3 patients were chosen exemplarily. Training sites from physiological and cancerous tissues were marked. Hyperspectral data from 1 patient were used to train a classifier, which was then used for automatic detection of precancerous and cancerous lesions in another 2 patients. RESULTS Intraoperative hyperspectral imaging was performed without any problems. Classification showed sensitivities of 61 and 43%, and a specificity of 100%. CONCLUSION This proof-of-concept study underscores the high potential of hyperspectral imaging for early recognition of cancer in the mouth and oropharynx. Besides a better prognosis for cancer patients, this approach could lead to higher cost efficiency in the health system.