Most patients with epidermolysis bullosa acquisita develop an autoimmune response to the non-collagenous (NC) 1 domain of type VII collagen. We report a 4-year-old girl of white European descent presenting with widespread blistering disease involving the face, hands, genital area and oral mucosa. Histopathology revealed subepidermal blisters, and linear deposits of IgG and C3 were seen along the dermal-epidermal junction on direct immunofluorescence (IF) microscopy of a perilesional skin biopsy. On indirect IF microscopy, circulating autoantibodies exclusively stained the dermal side of 1 mol L-1 NaCl-split skin. The patient's IgG autoantibodies labelled a 290-kDa protein on Western blotting of dermal extracts, and reacted with the NC1, NC2 and triple helical domains of type VII collagen on immunoblotting of recombinant and cell-derived fragments obtained by pepsin and collagenase digestion of the full-length protein. Oral methylprednisolone and dapsone led to clearance of lesions, which healed with mild scarring and milia formation. Treatment was discontinued after 1 year and the patient has now been in remission for more than 3 years.

Physical examination and ultrasound B-scan screening are important follow-up procedures in melanoma patients with regional disease. However, they do not allow definite diagnosis of suspicious lesions. Fine-needle aspiration cytology (FNAC) enhances the diagnostic accuracy in such patients but, unfortunately, reaches its technical limits, particularly when very small or necrotic lesions are examined. We therefore tested whether tyrosinase reverse transcription polymerase chain reaction (RT-PCR) of fine-needle aspirates (FNA-PCR) could help to increase diagnostic sensitivity. With clinical follow-up in 69 melanoma patients 81 regional lymph nodes were detected by ultrasound B-scan examination, nine of whom appeared to be palpable. Technically, FNAC was successful in all 81 lymph nodes, while FNA-PCR failed to obtain RNA at detectable levels in two lymph nodes of two patients. Of 79 lesions which have been completely evaluated by B-scan, FNAC and FNA-PCR, 44 proved to be melanoma metastases by histopathology, while the remaining 35 lesions were finally classified as non-specific lymph nodes. Of the 44 melanoma metastases 80% (n = 35) have been detected by B-scan, 90% (n = 39) by FNAC and 100% (n = 44) by FNA-PCR (P < 0.05 vs FNAC, P < 0.005 vs B-scan). In the subclass of lesions with diameters below 10 mm the sensitivities were 72% (n = 13), 78% (n = 14) and 100% (n = 18) respectively. In 35 regional lymph nodes classified as benign lesions, FNAC was always negative while FNA-PCR produced one positive result. Neither of these methods did produce false positive results in 15 control lymph nodes of non-melanoma patients. We conclude, that FNA-PCR might have superior sensitivity as compared to FNAC or ultrasound B-scan, particularly in melanoma lesions with diameters below 10 mm.

The clinical value of the reverse transcription polymerase chain reaction (RT-PCR) assay for tyrosinase in peripheral blood of melanoma patients is still under debate. A total of 212 blood samples from 212 melanoma patients in all clinical stages (AJCC) were examined. Erythrocytes were lysed prior to RNA extraction by phenol precipitation from 2.7 ml of blood. cDNA for tyrosinase PCR was synthesized using random hexamers. Positive tyrosinase RT-PCR results were obtained in 11% of 106 stage I patients, 18% of 56 stage II patients, 31% of 26 stage III patients and 67% of 24 stage IV patients. After a median follow-up of 36 months (range 26–41), stage III patients with positive RT-PCR for tyrosinase had a shortened disease-free interval as compared to negative patients (P< 0.01). In stage IV patients, median overall survival was 8 months in case of a positive RT-PCR in contrast to 12 months in case of a negative test. While univariate analysis showed sex and primary tumour location associated with positive RT-PCR, multiple regression analysis revealed clinical stage and detection of tyrosinase transcripts in peripheral blood as best prognostic factors. Hazard ratios for disease-free survival were 19.7 (confidence interval (CI) 8.53–45.5, P = 0.0001) for metastatic vs primary disease and 2.96 (Cl 1.49–5.89, P = 0.002) for positive vs negative tyrosinase RT-PCR. The corresponding hazard ratios for overall survival were 97.0 (Cl 12.7–741, P = 0.0001) and 4.33 (Cl 1.69–11.1, P = 0.002). Our results emphasize the importance of tyrosinase RT-PCR testing in peripheral blood. © 2000 Cancer Research Campaign

Lasers in Surgery and MedicineVolume 23, Issue 5 p. 249-249 Letter to the Editor Boiling and splattering liquid with the Er:YAG laser† B. L. Ziegler, B. L. Ziegler Department of Hematology and Oncology, Eberhard-Karl-University of Tübingen, D-72076 Tübingen, GermanySearch for more papers by this authorC. A. Thomas, C. A. Thomas Department of Physiology and Cellular Biophysics, Columbia University, New York, New York 10032Search for more papers by this authorT. Meier, T. Meier Institute of Laser Technologies in Medicine, D-89070 Ulm, GermanySearch for more papers by this authorR. Müller, R. Müller Department of Clinical Psychology and Occupational Medicine, University of Ulm, D-89070 Ulm, GermanySearch for more papers by this authorT. M. Fliedner, T. M. Fliedner Department of Clinical Psychology and Occupational Medicine, University of Ulm, D-89070 Ulm, GermanySearch for more papers by this authorL. Weber, L. Weber Department of Clinical Psychology and Occupational Medicine, University of Ulm, D-89070 Ulm, GermanySearch for more papers by this author B. L. Ziegler, B. L. Ziegler Department of Hematology and Oncology, Eberhard-Karl-University of Tübingen, D-72076 Tübingen, GermanySearch for more papers by this authorC. A. Thomas, C. A. Thomas Department of Physiology and Cellular Biophysics, Columbia University, New York, New York 10032Search for more papers by this authorT. Meier, T. Meier Institute of Laser Technologies in Medicine, D-89070 Ulm, GermanySearch for more papers by this authorR. Müller, R. Müller Department of Clinical Psychology and Occupational Medicine, University of Ulm, D-89070 Ulm, GermanySearch for more papers by this authorT. M. Fliedner, T. M. Fliedner Department of Clinical Psychology and Occupational Medicine, University of Ulm, D-89070 Ulm, GermanySearch for more papers by this authorL. Weber, L. Weber Department of Clinical Psychology and Occupational Medicine, University of Ulm, D-89070 Ulm, GermanySearch for more papers by this author First published: 04 January 1999 https://doi.org/10.1002/(SICI)1096-9101(1998)23:5<249::AID-LSM3>3.0.CO;2-O † This is a reply to Dr. Hughes' Letter to the Editor, which was published in LSM 23:248 (1998). AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume23, Issue51998Pages 249-249 RelatedInformation

A novel model system was used to investigate the spread of infectious particles and live cells through the application of lasers commonly used in clinical medicine. Supernatants from a cell line producing recombinant retroviruses carrying a marker gene (neoR) were exposed to Er:YAG‐laser beams. Aerosols were collected from various sites and distances from the point of laser impact and were analyzed by reverse transcription‐polymerase chain reaction (RT‐PCR) for neoR. In addition, a susceptible indicator cell line was used to investigate the presence of infectious virions in collected aerosols. To test the possibility of dissemination of viable cells, a cell line was laser irradiated, and the generated aerosols were analyzed for the presence of viable cells. The viral marker gene neoR could be detected in 16% (distance: 5.0–6.3 cm) to 59% (0.5–1.6 cm) of wells adjacent to the point of laser impact. The presence of infectious viruses in laser vapors conferring G418 resistance could be detected in 3% (distance 5.0–6.3 cm) to 20% (distance: 0.5–1.6 cm) of wells containing susceptible cells, and subsequent PCR analysis of isolated resistant clones revealed the presence of neoR‐RNA and ‐DNA. Viable cells were detected in 40% (distance 0.7–3.6 cm) to 3% (distance 10.7–11.8 cm) of wells adjacent to the point of laser impact. These results demonstrate that laser vapors can contain infectious viruses, viral genes, or viable cells and may promote the spread of infections or tumor cell dissemination. Lasers Surg. Med. 22:37–41, 1998. © 1998 Wiley‐Liss, Inc.

Different types of porcine tissue were irradiated with a surgical CO2 laser. The generated aerosols were sampled on glass fiber filters and incubated with human peripheral blood cells. Afterwards, these exposed cells were subjected to the comet assay. The single cell gel electrophoresis or comet assay represents a powerful technique for the detection of DNA strand breaks in eukaryotic cells. In short, the electrophoretic mobility of DNA fragments is proportional to the quantity of DNA damage caused by the genotoxic agent in question. By investigating porcine tissue laser pyrolysis products, it is demonstrated that the comet assay is an appropriate tool to assess the genotoxic capacity of even a heterogeneously composed class of substances with unknown modes of action and interaction.

We report on investigations of laser ablation of larger particles which may be in the minority but are known to be the main carrier of possibly infectious materia. We determined the dynamics of the ablated material by means of short-time exposure video recording with a Schlieren-Optik device. In addition, ablated particles were collected on microscopical slides and examined visually. The region of interest was imaged on a CCD-camera and transferred to a image processing system to get information about the size distribution and the morphology of the particulate matter. For the experiments we irradiated both soft and hard tissues and tissue modelling substances with pulsed and, for comparison, with continuous wave lasers. The particle velocities and the morphology of the ablated matter, either of irregular or special form, depend strongly on the laser type and laser parameters, respectively the treated material. In case of spherical aerosols we quantitatively determined the particle size distributions, for irregular shaped particles (mostly fibrous) only qualitative statements can be made.

Porcine liver was irradiated with a medical CO2-laser. The generated particles were sampled with glass fiber filters and a clean-up procedure specific for polycyclic aromatic hydrocarbons (PAH) was carried out. Separation and detection was done by gas chromatography/mass spectrometry. This clean-up procedure was controlled by fluorinated Internal Standards. Rather low amounts of PAH and their guide substance Benzo[a]pyrene were found. Compared to its threshold limit value no potential risk for the medical staff seems to be given.

Schweineleber wurde mit einem medizinischen CO2-Laser bestrahlt. Die partikelförmigen Pyrolyseprodukte wurden mit Glasfaserfiltern gesammelt und spezifisch auf polycyclische aromatische Kohlenwasserstoffe (PAH) aufgearbeitet. Die Aufarbeitung wurde mit Hilfe fluorierter Interner Standards (IS) überprüft. Die gefundenen Gehalte an PAH sowie deren Leitsubstanz Benzo[a]pyren waren sehr gering und lagen deutlich unterhalb des Grenzwerts für Benzo[a]pyren am Arbeitsplatz.

Laser-tissue interaction may generate by energy absorption a complex mixture of gaseous, volatile, semi-volatile and particular substances. At the time about 150 different components are known from IR-laser interaction with different organ tissues like liver, fat, muscle and skin. The laser-tissue interaction process thereby is dominated by heating processes, which is confirmed by the similarity of formed chemical products in comparison with conventional cooking processes for food preparation. With the identified chemical substances and relative amounts in backmind a walk along the think path of risk assessment with special reference to pyrolysis products is given. The main way of intake of pyrolysis products is the inhalative one, which results from the fine aerosols formed and the high spreading energy out of the irradiated source. The liberated amounts of irritative chemicals as (unsaturated) aldehydes, heterocycles of bad odor and possibly cancerogenic acting substances relates to some (mu) g/g of laser vaporized tissue. With regard to this exposure level in a hypothetic one cubic meter volume the occupational limit settings are far away. Even indoor air exposure levels are in nearly all cases underwent, for the content of bad smelling substances forces an effective ventilation. Up to now no laser typical chemical substance could be identified, which was not elsewhere known by frying or baking processes of meat, food or familiar. Starting with the GRAS concept of 1957 the process of risk assessment by modified food products and new ingredients is still improofing. The same process of risk assessment is governing the laser pyrolysis products of mammalian tissues. By use of sufficient suction around the laser tissue source the odor problems as well as the toxicological problems could be solved.

Actual occupational infections of medical staff is dominated by HBV, HIV and HCV-infections by dermal blood inocculation like needle injuries. What amount of these blood borne infections was possibly done via the aerosol pathway is unknown today. Looking at the laser generated aerodynamic particle sizes and the particle size of humanpathogen viruses as circulating or cell fixed units shows common trans-mission abilities to the human respiratory system. In cell tissue monolayer model systems and contami-nated serum sytems with virus infections this mechanisms were demonstrated as viable. For safety evaluation the lifetime, spreading behaviour and infection potential by viruses and bacterias of contaminated human laser aerosol must be further characterized.

Different tissue samples have been irradiated with surgical XeCl- and CO2-lasers. The generated laser plume was sampled and analyzed concerning medium and low volatile organic compounds. Differences in the composition of the pyrolysis products in dependance of tissues and lasers are presented. Quantification of aromatic hydrocarbons was carried out. It is obvious that the ratios between the single aromatic hydrocarbons gave hints at the temperatures of the laser tissue interaction process. Some aromatic hydrocarbons were typical high temperature products like phenylacetylene, whereas toluene could be found at lower temperatures with comparable high concentration. Two special classes of compounds, presumed by Curie point pyrolysis of proteins and not yet verified by synthesis, were identified in the aerosol of the CO2-laser. Probably five different amino acids might be the precursors of these compounds whereas by Curie point pyrolysis only three amino acids were reported as precursors. The particular debris which was sampled separately on glass fiber filters was extracted with different solvents. Several compounds absorbed at the particles could be identified and will be discussed. In the polar acetone extract some of main compounds remained unknown. A special clean-up procedure for polycyclic aromatic hydrocarbons (PAH) was carried out. Qualitative and quantitative results of the PAH analysis are presented. The results are compared with the results of other working groups.

Laser plume and particulate debris distribution during laser ablation of tissue can vary strongly depending on laser type and laser parameters. Investigations were made by means of particle flow visualization and particle density monitoring for both cw- and pulsed lasers. It turns out that the dynamics of fine dust particles are quite different from those of larger particles consisting of aggregates of cell fragments or complete cells. Consequences for the design of plume suction units and other safety means are discussed.

Use of laser systems in minimal invasive surgery results in formation of laser aerosol with volatile organic compounds of possible health risk. By use of currently identified chemical substances an overview on possibly associated risks to human health is given. The class of the different identified alkylnitriles seem to be a laser specific toxicological problem. Other groups of chemicals belong to the Maillard reaction type, the fatty acid pyrolysis type, or even the thermally activated chemolysis. In relation to the available different threshold limit values the possible exposure ranges of identified substances are discussed. A rough estimation results in an exposure range of less than 1/100 for almost all substances with given human threshold limit values without regard of possible interactions. For most identified alkylnitriles, alkenes, and heterocycles no threshold limit values are given for lack of, until now, practical purposes. Pyrolysis of anaesthetized organs with isoflurane gave no hints for additional pyrolysis products by fragment interactions with resulting VOCs. Measurements of pyrolysis gases resulted in detection of small amounts of NO additionally with NO2 formation at plasma status.

Liver and muscle tissue have been irradiated with a surgical CO2-laser. The prefiltered fumes were adsorbed on different sorbents (activated charcoal type NIOSH and Carbotrap) and desorbed with different solvents (carbondisulphide and acetone). Analysis was done by gas chromatography/mass spectrometry. An updated list of identified substances is shown. Typical Maillard reaction products as found in warmed over flavour as aldehydes, aromatics, heterocyclic and sulphur compounds were detected. Quantification of some toxicological relevant substances is presented. The amounts of these substances are given in relation to the laser parameters and different tissues for further toxicological assessment.

Medical laser treatment enlarged its application in recent years in an explosive way. By a given distance to the patient the laser surgeon can cut, coagulate, or evaporate human tissue in a very distinct manner. Due to the mainly thermal interaction of the laser light with the irradiated tissue it may be heated up to pyrolysis conditions. By pyrolysis of human tissue degradation products are generated, which may be harmful. Chemical substances and particles formed of tissue could be toxic, cancerogenic or irritant to skin and airways or after uptake. Special hazards of human laser plume in the health care environment may result from infectious viruses, bacterias, parasites, spread tumor cells and DNA fragments.

Various tissue samples have been irradiated with lasers generally used for surgical laser applications. Laser generated fumes were collected on charcoal tubes and chemical analysis of the pyrolytic products was performed by means of gaschromatographic and mass spectrometric (GC/MS) methods. First experimental results show clearly distinguishable features in the component spectra from various combinations of tissues and lasers or laser parameters, respectively. A method of standardization and calibration is presented and preliminary estimations of emission concentrations of particular substances are given.

The collecting efficiency of surgical smoke evacuators depends strongly on the design of the suction nozzle. In order to study the flow conditions at the nozzle entrance we developed methods to visualize the flow in the surrounding area. The experimental set-up for visualization and recording of the streamlines is described. Flow patterns recorded on a video recorder allow us to estimate the efficiency of a particular nozzle design under various experimental conditions. The turn-over from laminar flow to turbulent flow due to the laser generated fume can be studied in detail.

We have characterized the 38‐kDa transformation‐associated membrane glycoprotein MH 99, whose expression is highly elevated in many epithelial malignancies. A spontaneous cleavage of MH 99 into a 32‐ and a 6‐kDa chain in some carcinoma cell lines was recently shown. Sequence homologies to nidogen, a matrix‐adhesion molecule, support the suggestion of a receptor‐like function. In this study, we characterized biochemical and immunogenic aspects of MH 99. Transformed epithelial cell lines which do not spontaneously cleave MH 99 were exposed to 8 proteases with distinct specificities. Each of the enzymes produced similar specific fragmentation into chains of about 30 to 32 and 6 kDa, indicating a characteristic cleavage site of MH 99. The fragments were not distinguishable from those in carcinoma cells showing spontaneous cleavage of MH 99. The specific fragmentation depends on the localization in intact membranes and is not shared by other membrane proteins. N‐glycosylation of MH 99 of about 4 kDa was exclusively found on the 32‐kDa fragment. Characterization of antigenic epitopes was performed using 16 different monoclonal antibodies (MAbs). Only 2 independent determinants were found. One is located on the 32‐kDa chain and is recognized only by the MM 104 MAb. The other 15 antibodies bind to a dominant epitope on the 6‐kDa fragment which can be divided into 3 overlapping sub‐epitopes. The unique features of MH 99 indicate that its immunogenic epitopes are mainly located at its 6‐kDa chain, and support the suggestion of a transformation‐associated cell‐surface receptor which might be proteolytically regulated.