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Publications

Decalcification

Articular cartilage and synovial tissue

(Ref. DE-001)

BioSyntech Inc., Montréal, Canada
E. Rossomacha et al.
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Standard histoprocessing for hard tissue or sensitive soft tissue typically involves a lengthy manual process. Advances in microwave (MW) technology offer new opportunities to facilitate histoprocessing without compromising results. In 1998, a new one-step dehydration/clearing agent called JFC solution (Milestone, Italy) was developed specifically for use in MW histoprocessing. JFC solution consists of absolute ethanol, and isopropyl alcohol with long-chain hydrocarbons and has transformed histoproccesing into a 2-step process: JFC and paraffin, and effectively reduced processing time to between 30 minutes and 3 hrs (depending on size and nature of tissue). Here we compared the histological quality of rabbit femoral articular cartilage and patellar synovial tissue following histoprocessing using two different methods: a) routine manual and b) MW processing with JFC solution for dehydration and clearing in one step.

Effect of microwave fixation and decalcification on rodent tissue

(Ref. DE-002)

Journal of Histotechnology
April L. Marr and Anthony Wong
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The histological use of laboratory microwaves was proposed by Mayers in 1970. Since then, microwaves have become widely used in histology laboratories for fixation, decalcification, and processing; however, protocols must be carefully optimized to ensure the maintenance of optimal cellular morphology and antigenicity. In the current study, we sought to decrease laboratory turnaround time and optimize specimen preservation conditions by using microwave fixation. Mouse and rat tissues were harvested and divided into two groups (A and B). Tissues in group A were fixed for 24 h in 10% neutral buffered formalin (NBF), whereas tissues in group B were fixed for 2 h in 10% NBF before being microwaved at 50°C for varying times. Tissues from the two groups were compared by a pathologist. We found that the optimal microwave formalin-fixation times necessary to preserve morphology were 29 min for mouse tissues and 30 min for rat tissues. These times allowed specimens to be placed on a processor 3 h after tissue collection and processed for embedding the following day. We also determined optimal times for microwave decalcification of mouse and rat sternums and femurs. Decalcification of rat femurs with the combined use of microwave fixation and decalcification saved nearly 2 days of specimen turnaround times. To assess the effect of microwave fixation on antigenicity, rat intestine samples from groups A and B were stained with Ki-67 and caspase 3 antibodies. When compared with tissues that had been bench-fixed for 24 h, microwave-fixed tissues showed no loss of antigenicity. We conclude that rodent fixation and decalcification times can be considerably reduced with the aid of a microwave, thereby decreasing study turnaround times, minimizing costs, and improving efficiency. Microwave fixation would be a valuable investment for any laboratory seeking to save time while maintaining optimal cellular morphology and antigenicity.

Analysis of the effect of various decalcification agents on the quantity and quality of nucleic acid recovered from bone biopsies

(Ref. DE-003)

Annals of Diagnostic Pathology
Veena M. Singh et al.
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Molecular studies are part of standard care for cancer patients. Bone, a common and sometimes sole site of metastasis, requires decalcification for morphological examination. Many commonly used decalcification agents contain strong acids that degrade nucleic acids. The paradigm shift in oncology, with biomarker targeted therapy and gene expression profiling analysis, requires sufficient nucleic acid recovery from bone biopsy specimens. We systematically studied the effects of a spectrum of decalcification agents on the quantity and quality of RNA and DNA recovered from bone biopsies. Multiple bone biopsies of similar size and cellularity were fixed in 10% neutral-buffered formalin, randomized to various decalcification agents for 2 hours then processed, and embedded. Tissue lysates were obtained from unstained sections and nucleic acid isolated. DNA and RNA were quantified. Assessment of DNA and RNA integrity was accomplished by comparison of the average cycle threshold by polymerase chain reaction of selected housekeeping genes for each agent. Results were then analyzed by 2-sample t test. There was a significant decrease in both DNA and RNA yield and integrity with strong acids (hydrochloric, nitric) vs 14% EDTA and formic acid. DNA yield was (mean nanograms) 6.15 vs 68.68 (P<.001) and RNA was (mean nanograms) 121.53 vs 288.89 (P=.003), respectively. DNA integrity (mean cycle threshold) was 35.79 vs 30.16 (P<.001), and RNA was 33.03 vs 26.5 (P<.001), respectively. Decalcification of bone biopsies with EDTA or formic acid agents was associated with a significant improvement in recovered nucleic acid quantity and quality.

Rapid processing of bone marrow biopsies (BMB’s) with excellent morphology, immunohistochemistry and DNA quality

(Ref. DE-004)

Department of Pathology, Radboud University Medical Centre, Nijmegen, the Netherlands
I. de Laak–de Vries et al.
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BMB is an essential part of the diagnosis in patients with haematological diseases. In line with other oncological fast diagnostic tracks we optimized the processing of BMB’s to achieve reporting the following day.

EDTA-based decalcification of bone and bone Marrow. Ideal tool for protein and nucleic acid preservation. A pilot study

(Ref. DE-005)

Memorial Sloan-Kettering Cancer Center, New York, USA
Peter Ntiamoah et al.
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Bone and bone marrow biopsies are performed to diagnose diseases such as hematologic malignancies, primary bone tumors and metastatic tumors. In the era of increasing molecular testing for diagnosis and targeted therapeutics, preservation of nucleic acids and proteins are necessary and is required for the best patient care. For routine processing of bone specimens decalcification with inorganic acids (nitric acid or HCl) is used. Acid based methods allow fast turnaround times, but, the procedure restricts use of the tissue for molecular diagnosis by damaging DNA, RNA and is not suitable for some protein assays. To address this we undertook a pilot study using 10% EDTA decalcification solution, pH 7.2, as an alternate source for decalcification.

How we do: optimizing bone marrow biopsy logistics for sign-out within 2 days

(Ref. DE-006)

Journal of Hematopathology
I. de Laak–de Vries et al.
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Since the introduction of fast diagnostic tracks in many areas of oncology, the traditional processing of bone marrow biopsies (BMB), requiring either resin embedding or lengthy fixation and decalcification, is due to an upgrade. Thanks to a growing number of new commercially available tissue processors, microwave-enhanced processing is becoming a standard tool in the pathology laboratory, allowing rapid fixation and decalcification of BMB with preserved morphology and antigens. In this short report, we describe the use of a commercially available EDTA-based decalcification fluid (USEDECALC, Medite, Orlando, USA) in combination with the LOGOS J (Milestone, Bergamo, Italy), a closed microwave-enhanced tissue processor, for overnight fixation, decalcification, and paraffin impregnation of the BMB. This allows next-day reporting without impaired morphology or immunohistochemistry, and even improved DNA quality of the BMB.

Influence of decalcification procedures on immunohistochemistry and molecular pathology in breast cancer

(Ref. DE-007)

Modern Pathology
Willemijne AME Schrijver et al.
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Distant breast cancer metastases are nowadays routinely biopsied to reassess receptor status and to isolate DNA for sequencing of druggable targets. Bone metastases are the most frequent subgroup. Decalcification procedures may negatively affect antigenicity and DNA quality. We therefore evaluated the effect of several decalcification procedures on receptor status and DNA/RNA quality. In 23 prospectively collected breast tumors, we compared ER?, PR and HER2 status by immunohistochemistry in (non-decalcified) tissue routinely processed for diagnostic purposes and in parallel tissue decalcified in Christensen’s buffer with and without microwave, EDTA and Formical-4. Furthermore, HER2 fluorescence in situ hybridization and DNA/RNA quantity and quality were assessed. We found that the percentage of ER?-positive cells were on average lower in EDTA (P=0.049) and Formical-4 (P=0.047) treated cases, compared with controls, and PR expression showed decreased antigenicity after Christensen’s buffer treatment (P=0.041). Overall, a good concordance (weighted kappa) was seen for ER?, PR and HER2 immunohistochemistry when comparing the non-decalcified control tissues with the decalcified tissues. For two patients (9%), there was a potential influence on therapeutic decision making with regard to hormonal therapy or HER2-targeted therapy. HER2 fluorescence in situ hybridization interpretation was seriously hampered by Christensen’s buffer and Formical-4, and DNA/RNA quantity and quality were decreased after all four decalcification procedures. Validation on paired primary breast tumor specimens and EDTA-treated bone metastases showed that immunohistochemistry and fluorescence in situ hybridization were well assessable and DNA and RNA yield and quality were sufficient. With this, we conclude that common decalcification procedures have only a modest negative influence on hormone and HER2 receptor immunohistochemistry in breast cancer. However, they may seriously affect DNA/RNA-based diagnostic procedures. Overall, EDTA-based decalcification is therefore to be preferred as it best allows fluorescence in situ hybridization and DNA/RNA isolation.

Optimization of nasal tissue decalcification technology in preclinical studies of inhaled drugs: applied to rat nasal mucosa tissue Pathological examination

(Ref. DE-008)

Chinese Journal of Modern Drug Application
Wang Yu et al.
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OBJECTIVE: New inhaled formulations that act on the nose, mouth, respiratory tract, and whole body have received increasing attention. Meanwhile, the research and declaration of inhaled drugs have become hot spots amid infectious respiratory pandemic diseases worldwide. Due to the special anatomic structure of the nose, folds, grooves, and special structures may cause the specific uptake and deposition of inhaled substances. There are various epithelial tissues, glands, muscles, and cartilages in the vestibule, respiratory, and olfactory parts of the nose. Inhaled substances can generate irritating and toxic effects on various parts. The pathological diagnosis results from the preclinical safety evaluation of inhaled drugs are considered the gold standard for judging drug toxicology. The nose is composed of many bone components, and decalcification is required for the sectioning of hard bone tissues. Therefore, an efficient and high-quality decalcification method is the crucial pathological technique for evaluating inhaled drugs. METHODS: In this study, 10% ethylenediamine tetraacetic acid(EDTA), 10% formic acid, and 5% nitric acid decalcification solutions were selected. Besides, the decalcification time and effect of these decalcification solutions for rat nasal tissues were compared and analyzed under static room temperature and microwave conditions. Moreover, the quality of pathological bone tissue sections prepared through different decalcification methods was comprehensively evaluated. RESULTS:Compared with the decalcification method under normal temperature, the decalcification time under the treatment of KOS decreased significantly. The treatment with the EDTA decalcification solution had the longest decalcification time under normal temperature, while the treatment with the nitric acid decalcification solution had the shortest decalcification time under microwaves. During section evaluation, the EDTA decalcification solution had a higher quality score under normal temperature and microwaves, which indicated that the section quality was favorable. The nitric acid decalcification solution had a lower section quality score under microwaves, which indicated that the section quality was unfavorable. There was medium section quality for the formic acid decalcification solution under microwaves and normal temperature and for the nitric acid decalcification solution under normal temperature. The HE staining results suggested that there were incomplete nasal mucosa epithelia, fragmentation, and pink nasal bone tissues in the tissue sections treated by the nitric acid decalcification solution, presenting a peracid state. In the tissue sections treated by the formic acid decalcification solution and the EDTA decalcification solution, the nucleus of epithelial cells was blue-purple, the cytoplasm and interstitial components were pink, and the epithelial tissue structure of nasal mucosa was intact. The MASSON staining results suggested that in the tissue sections treated by the nitric acid decalcification solution, the whole section staining was red, the positive area was not obvious, and the epithelial cell differentiation was not prominent, with a fuzzy structure. In the tissue sections treated by the formic acid decalcification solution, the sections were slightly detached during staining, and slight cracks were observed in submucosa tissues. In the tissue sections treated by the EDTA decalcification solution, the structure of positive regions and epithelial mucosa regions was clear, and the nuclear and interstitial components were clearly distinguished. The immunohistochemical staining (Ki67) results suggested that in the tissue sections treated by the nitric acid decalcification solution, the staining of positive regions was uneven, and there were nonspecific negative reactions in some regions. In addition, local epithelial cells were unstained. In the tissue sections treated by the formic acid decalcification solution, the local regions were not clearly stained, and nonspecific negative and positive reactions appeared in some local regions. In the tissue sections treated by the EDTA decalcification solution, the positive regions were prominent, the boundaries between negative regions and positive ones were clear, and each region of the sections was stained evenly. CONCLUSION: Among the three decalcification solutions in this study, the nitric acid decalcification solution had the shortest decalcification time while the poor section and staining quality. The decalcification time of nasal tissues through the EDTA decalcification solution combined with microwaves was significantly shorter than that through the EDTA decalcification solution at normal temperature. Furthermore, this decalcification method achieved favorable section and staining quality.

Fixation

Heat-accelerated fixation and rapid dissection of the pediatric brain at autopsy: a pragmatic approach to the difficulties of organ retention

(Ref. FI-001)

Pediatric and Developmental Pathology
Ciara Barrett et al.
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We investigated whether it is possible to accelerate the examination of a pediatric brain at autopsy and thus facilitate its return to the body before a funeral without compromising the quality of the neuropathologic examination. Accelerated fixation and next-day dissection of the brain was performed in selected cases over a 2-year period by using a microwave histologic tissue processor (MicroMed T/T MEGA, Milestone, Sorisole, Italy). Direct comparison of the histologic appearance and immunohistochemical reactivity of 2 cases, 1 fixed by conventional methods and 1 fixed with the accelerated method, was performed in a blinded fashion by a specialist neuropathologist. Examination of rapidly fixed brain by conventional thin coronal sections was readily achieved. There was no appreciable difference between tissue sections stained with hematoxylin and eosin and prepared from conventional formalin-fixed cortical and cerebellar brain tissue and that fixed by rapid heat acceleration. Immunocytochemical studies were not adversely affected by the accelerated heat-fixation process of tissue. Heat-accelerated fixation is a potential method of speeding up the examination of the brain at autopsy without unduly compromising the quality of the neuropathologic examination.

Improving placental block morphology using microwave-assisted fixation

(Ref. FI-002)

Journal of Histotechnology
Anthony Henwood
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The histopathologic assessment of placenta is often marred by poorly fixed and processed sections. An audit conducted in the department resulted in 21% of placental blocks showing features of poor processing. A microwave-assisted fixation procedure was introduced using the Micromed KOS microwave oven (ABACUS-ALS, Brisbane, Australia) for 2 h at 45°C. This resulted in an improvement in the quality of the sections with a zero failure rate on a subsequent audit.

Cold formalin fixation guarantees DNA integrity in formalin fixed paraffin embedded tissues: premises for a better quality of diagnostic and experimental pathology with a specific impact on breast cancer

(Ref. FI-003)

Frontiers in oncology
Enrico Berrino et al.
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Formalin fixation and paraffin embedding (FFPE) represent the standard method to preserve tissue specimens for diagnostic pathology, however formalin fixation induces severe fragmentation of nucleic acids. We investigated whether formalin fixation at 4°C could preserve DNA integrity in FFPE specimens. Paired samples from 38 specimens were formalin fixed at room temperature (stdFFPE) and at 4°C (coldFFPE), respectively. Two independent cohorts were prospectively collected, cohort A (collected 6 years prior to the study, n = 21), cohort B (collected at time of the study, n = 17). DNA was extracted and its integrity evaluated with a qPCR-based assay that produces a normalized integrity index, the QC score (ratio between the quantity of a long and a short amplicon of the same gene). We observed higher QC scores in coldFFPE compared to stdFFPE samples (mean values: 0.69 vs. 0.36, p < 0.0001) and stdFFPE breast cancer specimens showed the most detrimental effect overall. Comparable QC scores were obtained between coldFFPE tissues of both cohorts; conversely, DNA integrity of stdFFPE was significantly lower in cohort A compared to cohort B (p < 0.0001). Of note, QC scores of stdFFPE (but not of coldFFPE) samples were significantly reduced following 6 months of storage (p = 0.0001). Monitored formalin fixation at 4°C outperforms standard fixation in ensuring high-quality DNA, which is key to feasibility of downstream high-throughput molecular analyses. An important effect was observed over storage time, thus suggesting a likely better preservation of archival samples when this cold fixation protocol is used.

Frozen Sections

Freezing skin tissue in Mohs Micrographic Surgery using PrestoCHILL

(Ref. FS-001)

Department of pathology, Department of dermathology, Cruces University Hospital, Barakaldo, Spain
Veronica Caamaño Villaverde et al.
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Mohs micrographic surgery is distinguished by histologic examination of the complete surgical margin prepared from fresh frozen tissue. The purpose is to achieve the examination in one section of the undersurface, the sidewall, and the epidermal margin. To do so, the specimen must be manipulated before freezing, flattening it.

Mohs and the benefits of new embedding and staining systems

(Ref. FS-002)

Pathology in Practice
Guy Orchards
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Guy Orchards reports on preliminary work to assess the benefits of the latest equipment designed to facilitate frozen section preparation and staining in a laboratory supporting a Mohs micrographic surgery service.

Assessment of biopsies of donor kidneys prior to renal transplant. Comparison and validity of different freezing methods

(Ref. FS-003)

Dep. of Pathological Anatomy. Hospital Universitario Fundación Jiménez Díaz, Madrid, Spain
Ana Márquez Lillo et al.
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Histological evaluation of donor kidneys prior to transplantation is a mandatory practice in up to 60% of kidney transplants. There are several published scores that assess the following parameters: glomeruli, tubules, interstitium, arteries and arterioles. Microwave accelerated paraffin processors are not available in most centers and the sample is frozen for the study. Artifacts that occur in tissue in many cases prevents a proper assessment.

Comparación del sistema PrestoCHILL frente al de congelación en criostato, en el estudio de la biopsia renal

(Ref. FS-004)

Servicio de Anatomía Patológica. Hospital General Universitari de Castelló, Spain
M. Manasé et al.
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La técnica de inmunofluorescencia directa, que se realiza sobre tejido congelado, es fundamental en el estudio de la biopsia renal. Para su correcta evaluación es necesario un buen corte, con el máximo número de glomérulos íntegros. Recientemente se ha introducido un nuevo método basado en la congelación rápida de la muestra mediante un motor tipo Stirling refrigerado con gas helio que reduce la formación de cristales de hielo en el tejido. Además el posicionamiento invertido de la técnica permite un mayor control de primer nivel de corte.

Next Generation Frozen Sections – Hochwertige Gefrierschnitte durch Face-down Technologie

(Ref. FS-005)

Dept. of Pathology, Klinikum Bamberg, Germany
Gerhard Seitz
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Die Schnellschnittuntersuchung spielt eine zentrale Rolle in der Therapie und ist auch in vielen Situationen fur die optimale Therapie unentbehrlich, wenngleich in den letzten Jahren die Zahlen rucklaufig sind, da durch die vielfaltigen Methoden der Punktionstechniken die Dignitata der zu operierenden Lasionoftmals bereits praoperativ feststeht. Auf Homepages deutscher Institute und Praxen fur Pathologie sowie von Universitatskliniken wird unisono darauf hingewiesen, dass der Nachteil der Methode in einer etwas schlechteren Beurteilbarkeit des Gewebes besteht.Deshalb sei zur Diagnosesicherung etwa von Lymphomen eine Gefrierschnittuntersuchung kontraindiziert, da nach anschliebender Fixierung und Paraffineinbettung viele Schrumpfungsartefakte nachzuweisen sind. Die wesentlichen Indikationen fur Schnellschnittuntersuchungen sind heute – die Uberprufung eines chirurgischen Reseketionsrandes hinsichtlich der Tumorfreiheit, – die Unterscheidung zwischen gutund bosartigen Knoten der Schilddruse und bei Lungentumoren mit langsamen Watchstum, – die Bestimmung der Qualitat der Gewebeprobe (liegt ausreichendes und reprasentatives Gewebe fur eine definitive Diagnose vor?) sowie – die Untersuchung des Gewebes zur Auswahl fur spezielle Untersuchungen (molekulare Analysen, PCR etc.).

Qué son y cómo se procesan las biopsias?

(Ref. FS-006)

El Mercurio
Equipo de médicos anatomopatólogos del Instituto Oncológico FALP, Providencia, Cile
Read abstract…
El análisis de muestras de tejidos es una herramienta clave para confirmar o descartar la presencia de células malignas y también puede ayudar a definir un tratamiento personalizado. Los equipamientos para realizarlo son cada vez más sofisticados.

Telepathology Network in the Grand Duchy of Luxembourg Integrating a network of Hospitals with a centralized Pathology Laboratory

(Ref. FS-007)

Laboratoire National de Santé, Dudelange, Luxembourg
Val D. et al.
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Luxembourg is a country with five different hospitals and a single public Pathology Laboratory, the LaboratoireNational de Santé (LNS). This geographic separation poses challenges, the most notable of which is long delays in the pathologic intraoperative frozen section reporting process, since the fresh samples have to be sent to the LNS.

Validation of Remote Digital Frozen Sections for Cancer and Transplant Intraoperative Services

(Ref. FS-008)

Journal of Pathology Informatics
Luca Cima et al.
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Introduction: Whole?slide imaging (WSI) technology can be used for primary diagnosis and consultation, including intraoperative (IO) frozen section (FS). We aimed to implement and validate a digital system for the FS evaluation of cancer and transplant specimens following recommendations of the College of American Pathologists. Materials and Methods: FS cases were routinely scanned at ×20 employing the “Navigo” scanner system. IO diagnoses using glass versus digital slides after a 3?week washout period were recorded. Intraobserver concordance was evaluated using accuracy rate and kappa statistics. Feasibility of WSI diagnoses was assessed by the way of sensitivity, specificity, as well as positive and negative predictive values. Participants also completed a survey denoting scan time, time spent viewing cases, preference for glass versus WSI, image quality, interface experience, and any problems encountered. Results: Of the 125 cases submitted, 121 (436 slides) were successfully scanned including 93 oncological and 28 donor?organ FS biopsies. Four cases were excluded because of failed digitalization due to scanning problems or sample preparation artifacts. Full agreement between glass and digital?slide diagnosis was obtained in 90 of 93 (97%, ? = 0.96) oncology and in 24 of 28 (86%, ? = 0.91) transplant cases. There were two major and one minor discrepancy for cancer cases (sensitivity 100%, specificity 96%) and two major and two minor disagreements for transplant cases (sensitivity 96%, specificity 75%). Average scan and viewing/reporting time were 12 and 3 min for cancer cases, compared to 18 and 5 min for transplant cases. A high diagnostic comfort level among pathologists emerged from the survey. Conclusions: These data demonstrate that the “Navigo” digital WSI system can reliably support an IO FS service involving complicated cancer and transplant cases.

Cryoembedder, automatic processor-stainer, liquid nitrogen freezing, and manual staining for frozen sections examination. A comparative study

(Ref. FS-009)

Acta Histochemica
Salvatore Lorenzo Rennea et al.
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Frozen section examination (FSE) reshaped surgical pathology and is characterized by a high accuracy. Nonetheless pathologists can experience artefacts that can compromise or defer the diagnosis. We compared a commercial system, composed by a cryoembedder and a processor/stainer, to our FSE protocol. Feasibility of diagnosis as well as overall architecture, cytology, and staining, were scored under the following conditions: Traditional (liquid nitrogen freezing and manual staining), Only-Presto (liquid nitrogen freezing and commercial processor/stainer), Only-PrestoCHILL (cryoembedder and manual staining), and PrestoSystem (cryoembedder and processor/stainer). Scores were compared across the different experimental conditions. PrestoSystem had significantly higher scores than Traditional, Only-Presto or Only-PrestoCHILL in all categories (Wilcoxon test; all P-value<.001); similarly, also Only-Presto and Only-PrestoChill had significantly higher scores than Traditional in all categories. Only-PrestoCHILL had significantly higher scores than Only-Presto in Cytology and Architecture. In conclusion the control of pre-analytical variables provided reproducible results, of a higher quality.

NeuroSAFE in radical prostatectomy increases the rateof nerve-sparing surgery without affectingoncological outcome

(Ref. FS-010)

BJU International
Margaretha A. van der Slot et al.
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Objectives: To investigate the impact of intra-operative neurovascular structure-adjacent frozen-section examination (NeuroSAFE) onthe rate of nerve-sparing surgery (NSS) and oncological outcome in a large radical prostatectomy (RP) cohort.Patients and methods: Between January 2016 and December 2020, 1756 prostate cancer patients underwent robot-assisted RP, of whom 959 (55%)underwent this with NeuroSAFE and 797 (45%) without (control cohort). In cases where NeuroSAFE showed tumour inthe margin, a secondary resection was performed. The effect of NeuroSAFE on NSS and positive surgical margin (PSM)status was analysed using logistic regression. Cox regression was used to identify predictors of biochemical recurrence-freesurvival (BCRFS).Results and limitations: Patients in the NeuroSAFE cohort had a higher tumour grade (P<0.001) and clinical stage (P<0.001) than those in thecontrol cohort. NeuroSAFE enabled more frequent NSS for both pT2 (93% vs 76%;P<0.001) and pT3 disease (83% vs55%;P<0.001). In adjusted analysis, NeuroSAFE resulted in more frequent unilateral (odds ratio [OR] 3.90, 95%confidence interval (CI) 2.90–5.30;P<0.001) and bilateral (OR 5.22, 95% CI 3.90–6.98;P<0.001) NSS. While the PSMrate decreased from 51% to 42% in patients with pT3 stage disease (P=0.031), NeuroSAFE was not an independentpredictor of PSM status (OR 0.85, 95% CI 0.68–1.06;P=0.2) in the entire cohort. Patients who underwent NeuroSAFEhad better BCRFS compared to the control cohort (hazard ratio 0.62, 95% CI 0.45–0.84;P=0.002). This study is limited byits comparison with a historical cohort and lack of functional outcomes.Conclusions: NeuroSAFE enables more unilateral and bilateral NSS without negatively affecting surgical margin status and biochemicalrecurrence. This validation study provides a comprehensive overview of the implementation, evaluation and intra-operativedecision making associated with NeuroSAFE in clinical practice.

Test of the FlashFREEZE unit in tissue samples freezing for biobanking purposes

(Ref. FS-011)

Cell Tissue Bank
Edyta Biskup et al.
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Availability of molecularly intact biospecimens is essential in genetic diagnostics to obtain credible results. Integrity of nucleic acids (particularly RNA) may be compromised at various steps of tissue handling, and affected by factors such as time to freeze, freezing technique and storing temperature. At the same time, freezing and storing of the biological material should be feasible and safe for the operator. Here, we compared quality of DNA and RNA from biospecimens derived from different organs (breast, colon, adrenal glands, testes, rectum and uterus) frozen either using dry ice-cooled isopentane or with FlashFREEZE unit, in order to verify if the latter is suitable for routine use in biobanking. Implementing FlashFREEZE device would enable us to limit the use of isopentane, which is potentially toxic and environmentally harmful, whilst facilitate standardization of sample freezing time. We considered factors such RNA and DNA yield and purity. Furthermore, RNA integrity and RNA/DNA performance in routine analyses, such as qPCR, next generation sequencing or microarray, were also assessed. Our results indicate that freezing of tissue samples either with FlashFREEZE unit or isopentane ensures biological material with comparable expression profiles and DNA mutation status, indicating that RNA and DNA of similar quality can be extracted from both. Therefore, our findings support the use of the FlashFREEZE device in routine use for biobanking purposes.

Optimized whole-genome sequencing workflow for tumor diagnostics in routine pathology practice

(Ref. FS-012)

Nature Protocols
Kris G. Samsom et al.
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Two decades after the genomics revolution, oncology is rapidly transforming into a genome-driven discipline, yet routine cancer diagnostics is still mainly microscopy based, except for tumor type-specific predictive molecular tests. Pathology laboratories struggle to quickly validate and adopt biomarkers identified by genomics studies of new targeted therapies. Consequently, clinical implementation of newly approved biomarkers suffers substantial delays, leading to unequal patient access to these therapies. Whole-genome sequencing (WGS) can successfully address these challenges by providing a stable molecular diagnostic platform that allows detection of a multitude of genomic alterations in a single cost-efficient assay and facilitating rapid implementation, as well as by the development of new genomic biomarkers. Recently, the Whole-genome sequencing Implementation in standard Diagnostics for Every cancer patient (WIDE) study demonstrated that WGS is a feasible and clinically valid technique in routine clinical practice with a turnaround time of 11 workdays. As a result, WGS was successfully implemented at the Netherlands Cancer Institute as part of routine diagnostics in January 2021. The success of implementing WGS has relied on adhering to a comprehensive protocol including recording patient information, sample collection, shipment and storage logistics, sequencing data interpretation and reporting, integration into clinical decision-making and data usage. This protocol describes the use of fresh-frozen samples that are necessary for WGS but can be challenging to implement in pathology laboratories accustomed to using formalin-fixed paraffin-embedded samples. In addition, the protocol outlines key considerations to guide uptake of WGS in routine clinical care in hospitals worldwide.

Grossing

Image Documented Surgical Pathology (ID-SP) to enhance patient safety – Lean redesign of the value stream steps from gross examination to signout

(Ref. GR-001)

Department of Pathology, Here Henry Ford Health System, Detroit, Michigan, USA
Dhananjay A Chitale et al.
Read abstract…
Background: Mis-identification (Mis-ID) and Mis-comunication (Mis-COM) defects are frequent causes of potentially significant medical errors that threaten patient safety. The hand-off nature of surgical pathology processes from specimen collection to signout affordsopportunities for Mis-ID/Mis-COM failures. To further enhance safer surgical pathology, we have explored process and technologic innovations that leverage the maximum “a picture is worth a thousand words” by integrating ID-SP digital workstation employing images attached in the lab information system that visually documented all required information at gross examination to be used by downstream workstations. Design: Times to perform all steps of grossing and documentation were assessed comparing 25 biopsies grossed by a single pathology assistant with our traditional grossing protocol (TGP), dictationless & wordless, to ID-SP protocol developed for the LeanSTATION Bx digital macro-imaging system (Milestone Medical, Kalamazoo, MI). ID-SP documentation gross protocol consisted of sequential digital image recordings: 1-requisition, 2-specimen container label as received and barcoded cassette, 3- container contents as received, 4-tissue placed in cassette with superimposed 1 mm electronic measurung grid. Results: Average time required for ID-SP gross was 76 seconds per specimen vs. 37 seconds for TGP. Histology personnel could refer to gross specimen descriptions at time of embedding whereas actual images of submitted issues in cassettes were available attached to the case in Pathology PACS system (Apollo PACS, Inc, Falls Church, VA). These images where also availale to downstream histology personnel at cutting stations. Pathologists at the time of signout had additional visual quality control checks to confirm patient identification of requisitions,, container and cassette labels and submitted tissue. Conclusion: Grossing time of ID-SP protocol was double TGP, but the image protocol provided additional assurance of catching Mis_ID and Mis-COM errors with prospective visual quality conrol tracking of requisitions and specimens at each workstation from gross to pathologist. In our experience the time difference expended is more than recouped in the average rework time of 8 man-hours wasted in resolving a MIS-ID case searching for empty specimen containers, interviewing clinical & pathology personnel, performing DNA profiling and amending reports. Furteher efficiencies in some practices may be obtained with ID-SP work design by eliminating time & bottlenecks in trascription and report correction.

Photographic Documentation Support Of The Macroscopical Reporting In Anatomical Pathology: Impact To Histotechs’ Role

(Ref. GR-002)

U.O. Anatomia Patologica Ospedale “G. da Saliceto” di Piacenza, Italy
Manuela Ballarini and Elena Campiani
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Macroscopic reporting: It’s an essential step for the diagnostic process because it documents in writing the nature of the specimen under examination, describing: – Number of sent probes and their dimensions – Specimen’s shape and preservation modalityType of lesion – Potential surgical incisions – Samples’ orientation and potential special dying It’s an informal report enclosed to the case digital folder

Qué son y cómo se procesan las biopsias?

(Ref. GR-003)

El Mercurio
Equipo de médicos anatomopatólogos del Instituto Oncológico FALP, Providencia, Cile
Read abstract…
El análisis de muestras de tejidos es una herramienta clave para confirmar o descartar la presencia de células malignas y también puede ayudar a definir un tratamiento personalizado. Los equipamientos para realizarlo son cada vez más sofisticados.

Telepathology Network in the Grand Duchy of Luxembourg Integrating a network of Hospitals with a centralized Pathology Laboratory

(Ref. GR-004)

Laboratoire National de Santé, Dudelange, Luxembourg
Val D et al.
Read abstract…
Luxembourg is a country with five different hospitals and a single public Pathology Laboratory, the LaboratoireNational de Santé (LNS). This geographic separation poses challenges, the most notable of which is long delays in the pathologic intraoperative frozen section reporting process, since the fresh samples have to be sent to the LNS.

Large format histology – why thinner is better!

(Ref. GR-005)

National Society for Histotechnology
Dr. Philip Bryant et al.
Read abstract…
The importance of large format histology as a method of assessing wide areas of tissue samples in cancer pathology is unparalleled. For a pathologist to be able to examine tumor extent, involvement of surgical resection margins and other prognostic parameters in a complete cross section of the tissue on a single slide is unrivalled in cancer diagnostics. However, there are several issues that need to be addressed before attempting to process large format, supa mega (SM) tissue samples in the laboratory. In order to achieve optimal high-quality tissue processing combined with a reduction in processing times, the ability to gross tissues consistently to 5mm thickness is crucial. However, due to the countless types and variable consistencies of tissues, this is not always possible in the grossing room. To overcome these challenges, it is often helpful to first gross the tissue into thicker slices before placing them into 100% reagent alcohol or ethanol for an hour in order to stabilize the tissue. This treatment not only firms the tissue to allow thinner slices to be taken but often reduces the need for reprocessing fatty or other problematic tissue. If tissues contain large amounts of fat, placing them into a fat solvent such as acetone is advantageous before attempting to gross the tissue into thinner 5mm slices. In addition, using isopropanol as the dehydrating agent during processing of fatty tissue is often recommended as it is a superior fat solvent compared to ethanol. However, if reagent alcohol is your preferred choice as a dehydrating agent, then extending processing times in that reagent would be required to ensure optimal processing. Since minimizing the thickness of slices is essential for quality processing of large tissue samples, commercial aids are available for the consistent grossing of tissue. Systems such as the ProCUT slicing devices (Milestone Medical) and the TruSlice specimen cut up systems (CellPath) are not only convenient to use but also allow reliable production of thin slices of fresh and fixed tissues in the grossing room (Figures 1 & 2).

Macro Digital

Image is everything as MacroPATH arrives in the cut-up room

(Ref. MD-001)

Pathology in Practice
Azeem Hanif
Read abstract…
The istopathology cut-up room has changed little over time. Hpwever, with extended roles for biomedical scientists in this original laboratory specialty now at the pilot site stage, Surgipath Europe’s Azeem Hanif looks at a new digital system that will support both scientist and science.

Digital photography in anatomical pathology

(Ref. MD-002)

Postgraduate Medical Journal
Leong FJW-M and Leong ASY
Read abstract…
Digital imaging has made major inroads into the routine practice of anatomical pathology and replaces photographic prints and Kodachromes for reporting and conference purposes. More advanced systems coupled to computers allow greater versatility and speed of turnaround as well as lower costs of incorporating macroscopic and microscopic pictures into pathology reports and publications. Digital images allow transmission to remote sites via the Internet for consultation, quality assurance and educational purposes, and can be stored on and disseminated by CD-ROM. Total slide digitisation is now a reality and will replace glass slides to a large extent. Three-dimensional images of gross specimens can be assembled and posted on websites for interactive educational programmes. There are also applications in research, allowing more objective and automated quantitation of a variety of morphological and immunohistological parameters. Early reports indicate that medical vision systems are a reality and can provide for automated computer-generated histopathological diagnosis and quality assurance.

La tecnologia avanzata sbarca a Medicina legale. E gli esami vanno in…tv

(Ref. MD-003)

La Nazione, Italian National Newspaper
Cecilia Marzotti
Read abstract…
La tecnologia entra a pieno titolo nella Medicina legale senese. La novità è una piccola telecamera in grado di registrare immagini e parole durante un’autopsia che possono diventare un prezioso aiuto per il pubblico ministero nel corso di un processo e trasformarsi in informazioni preziosissime per la ricerca e l’archivio della setssa sezione di Scienze medico legali diretta dal professor Mario Gabbrielli che fa parte del dipartimento di patologia umana e oncologica medica il cui responsabile è la professoressa Marcella Cintorino. La medica legale dispone di una preziosa documentazione che poche altre in Italia vantano: l’archivio di oltre cento anni di esami autoptici. E lì c’è di tutto.

Guidelines for handling the most common and important surgical specimens

(Ref. MD-004)

Surgical Pathology
Rosai and Ackerman’s
Read abstract…
In order to achieve a certain consistency in the way the specimens are handled in the gross room, it is important for a manual of procedures to be available to the person performing the gross examination to assist in dissecting the specimen, describing it, taking the appropriate sections for microscopic examination, and performing whatever other addiotional tasks may be required depending on the nature of the case.

Educational Pathology: Tips and Tricks

(Ref. MD-005)

The journal of AAPA
Bill Ahlfeld
Read abstract…
A picture is worth a thousand words. This sentence has ended every column (including this one) since its inception. And what does it mean? Often, photography can say in a single picture, what would require a large explanation, or maybe couldn’t even be described clearly with words. So, how can we as PAs benefit most from this technology? While we are all accustomed to taking pictures of interesting surgical specimens, or those requested by surgeons or protocol, there are other benefits a photo can provide. We recently purchased the MacroPathD photo setup and software from Milestone.

Case Report of Autopsy and Placental Examination After Radiofrequency Ablation of an Acardiac Twin

(Ref. MD-006)

Lab Med Summer
Papreddy Kashireddy et al.
Read abstract…
We report the autopsy and placental findings in a monochorionic twin gestation complicated by twin reversed arterial perfusion (TRAP) sequence. Radiofrequency ablation (RFA) was performed at 24 weeks gestation to abort the acardiac fetus, and vaginal delivery of the co-twin and acardiac fetus occurred at 33 weeks gestation. An autopsy of the acardiac fetus revealed multiple congenital anomalies including complete absence of the upper extremities and poor development of the skull and facial structures. In contrast to the upper body, the lower half of the body, although malformed, was more developed. The monochorionic twin placenta showed velamentous, atrophied, proximal artery-artery and vein-vein intertwin vascular connections which essentially bypassed the placental parenchyma for the acardiac fetus. Ink injection and histologic examination confirmed thrombosis of these critical intertwin vascular connections after RFA. This report highlights the fetal and placental anatomy of TRAP sequence and stresses the importance of placental examination after fetal surgical techniques.

Image Documented Surgical Pathology (ID-SP) to enhance Patient Safety – Lean Redesign of the Value Stream Steps from Gross Examination to Signout

(Ref. MD-007)

Department of Pathology, Here Henry Ford Health System, Detroit, Michigan, USA
Dhananjay A Chitale et al.
Read abstract…
Background: Mis-identification (Mis-ID) and Mis-comunication (Mis-COM) defects are frequent causes of potentially significant medical errors that threaten patient safety. The hand-off nature of surgical pathology processes from specimen collection to signout affordsopportunities for Mis-ID/Mis-COM failures. To further enhance safer surgical pathology, we have explored process and technologic innovations that leverage the maximum “a picture is worth a thousand words” by integrating ID-SP digital workstation employing images attached in the lab information system that visually documented all required information at gross examination to be used by downstream workstations. Design: Times to perform all steps of grossing and documentation were assessed comparing 25 biopsies grossed by a single pathology assistant with our traditional grossing protocol (TGP), dictationless & wordless, to ID-SP protocol developed for the LeanSTATION Bx digital macro-imaging system (Milestone Medical, Kalamazoo, MI). ID-SP documentation gross protocol consisted of sequential digital image recordings: 1-requisition, 2-specimen container label as received and barcoded cassette, 3- container contents as received, 4-tissue placed in cassette with superimposed 1 mm electronic measurung grid. Results: Average time required for ID-SP gross was 76 seconds per specimen vs. 37 seconds for TGP. Histology personnel could refer to gross specimen descriptions at time of embedding whereas actual images of submitted issues in cassettes were available attached to the case in Pathology PACS system (Apollo PACS, Inc, Falls Church, VA). These images where also availale to downstream histology personnel at cutting stations. Pathologists at the time of signout had additional visual quality control checks to confirm patient identification of requisitions,, container and cassette labels and submitted tissue. Conclusion: Grossing time of ID-SP protocol was double TGP, but the image protocol provided additional assurance of catching Mis_ID and Mis-COM errors with prospective visual quality conrol tracking of requisitions and specimens at each workstation from gross to pathologist. In our experience the time difference expended is more than recouped in the average rework time of 8 man-hours wasted in resolving a MIS-ID case searching for empty specimen containers, interviewing clinical & pathology personnel, performing DNA profiling and amending reports. Furteher efficiencies in some practices may be obtained with ID-SP work design by eliminating time & bottlenecks in trascription and report correction.

Macro digital pathology imaging: advanced practice in Manchester

(Ref. MD-008)

Pathology in Practice
Caroline Glennie
Read abstract…
Caroline Glennie and colleagues at Manchester Royal Infirmary have been using the MacroPATH pro-x system from Menarini since January. Here, they report on a revolution in histopathology dissection.

Photographic Documentation Support Of The Macroscopical Reporting In Anatomical Pathology: Impact To Histotechs’ Role

(Ref. MD-009)

U.O. Anatomia Patologica Ospedale “G. da Saliceto” di Piacenza, Italy
Manuela Ballarini and Elena Campiani
Read abstract…
Macroscopic reporting: It’s an essential step for the diagnostic process because it documents in writing the nature of the specimen under examination, describing: – Number of sent probes and their dimensions – Specimen’s shape and preservation modalityType of lesion – Potential surgical incisions – Samples’ orientation and potential special dying It’s an informal report enclosed to the case digital folder

¿Qué son y cómo se procesan las biopsias?

(Ref. MD-010)

El Mercurio
Equipo de médicos anatomopatólogos del Instituto Oncológico FALP, Providencia, Cile
Read abstract…
El análisis de muestras de tejidos es una herramienta clave para confirmar o descartar la presencia de células malignas y también puede ayudar a definir un tratamiento personalizado. Los equipamientos para realizarlo son cada vez más sofisticados.

Telepathology Network in the Grand Duchy of Luxembourg Integrating a network of Hospitals with a centralized Pathology Laboratory

(Ref. MD-011)

Laboratoire National de Santé, Dudelange, Luxembourg
Val D et al.
Read abstract…
Luxembourg is a country with five different hospitals and a single public Pathology Laboratory, the LaboratoireNational de Santé (LNS). This geographic separation poses challenges, the most notable of which is long delays in the pathologic intraoperative frozen section reporting process, since the fresh samples have to be sent to the LNS. (Fig 1)

Telemacropatologia, una herramienta util en el trabajo diario del patologo. Nuestra experiencia.

(Ref. MD-012)

Laboratorios Ugalde Herrá & Asocs, Cuenca, Equador
Jorge Ugalde Puyol et al.
Read abstract…
La Anatomía patológica es estudio, análisis, evaluación y diagnóstico basados en las imágenes macro y micro de los especímenes. La necesidad de documentar el proceso de tallado de la pieza, realizado muchas veces por residentes, o técnicos, lo que impide al patólogo la valoración de parámetros importantes para el informe anatomopatológico, nos lleva a diseñar, con nuestros ingenieros electrónico, informático y bioinformático, en nuestro servicio, una mesa de tallado “ad hoc”, a la que después se une una Milestone Bx, y desarrollar una plataforma, protocolizando el uso diario de una herramienta de apoyo, remoto y presencial, útil en diagnóstico, docencia, revisión, morfometría y archivo de los procedimientos.

Processing

Ultra-Rapid Microwave/Variable Pressure-Induced Histoprocessing: Description of a New Tissue Processor

(Ref. PR-001)

The Journal of histotechnology
F.Visinoni et al.
Read abstract…
We describe a new method of ultra-rapid histoprocessing that reduces the processing times for needle and endoscopic biopsies to 30 min and that of other surgical biopsy tissue blocks of up to 4 mm thick to 120 min. The MicroMED U R M Histoprocessor, which combines microwave irradiation with precise computer control of power, time, temperature, vacuum, and pressure, when used with a 1-step dehydrating/clearing reagent, consistently produces rapidly processed tissues with optimal cytomorphology and improved tissue sectioning properties. Staining properties are excellent with no deleterious effects on routine staining, histochemistry, or immunohistochemistry. This new processing technique represents a major change from conventional tissue processing and eliminates the use of hazardous reagents such as xylene. The ease of application and speed of this technique can significantly reduce turnaround times in diagnostic laboratories.

Fast but not Furious

(Ref. PR-002)

Pathology In Practice
Sue Bennett
Read abstract…
Surgipath Europe product manager Sue Bennett has encountered some scepticism about the application of microwave technology in histopathology.But, she says, all it takes to melt away any misgivings is to see the RHS-1 process and then compare the results with conventionally prepared specimens.

Same-Day EM-Diagnosis of Tissues and Potential Bioterrorism Samples by the Use of Microwave-Assisted Processing

(Ref. PR-003)

Pathology Department , University Jospital, Regensburg, Germany
J.A. Schroeder et al.
Read abstract…
The purpose of this study was to compare the turnaround time as well as the section and image quality of specimens processed for electron microscopic diagnostic examination by microwave technology versus conventionally processed samples.

The fast-track biopsy (FTB): description of a rapid histology and immunohistochemistry method for evaluation of preoperative breast core biopsies

(Ref. PR-004)

International Journal of Surgical Pathology
Teresa Ragazzini et al.
Read abstract…
Thirty-six core breast biopsies from 32 patients were paraffin embedded by use of an automated microwave processor. In addition, a quick immunohistochemical method was used in selected cases. The quality of the hematoxylin and eosin (H&E) slides was very satisfactory, as were also the immunohistochemical stains for ER, PR, and Ki67 when compared to those obtained with the use of a conventional automated immunostainer. The time required to process the tissue to the final H&E stage averaged 2 hours 52 minutes, and the immunohistochemical method required 90 to 100 minutes. This procedure, which we named “fast-track biopsy” (FTB), is quick enough to be competitive with FNAC (fine-needle aspiration biopsy) in terms of turnaround-times. The superiority of core biopsy over FNA in terms of the morphologic information provided is widely acknowledged, the only major argument currently mentioned in favor of FNAC being the shorter duration of the procedure. With the advent of FTB, it would appear that even this last remaining advantage has been erased.

Microwaves in the Retrieval of Proteins, RNA and DNA

(Ref. PR-005)

Discipline of Anatomical Pathology, University of Newcastle, Australia
Anthony S-Y Leong
Read abstract…
Microwaves (MWs) are a form of non-ionizing radiation with a typically standard frequency of 2.45 GHz, a wavelength of 12.2 cm and photon energy of 10-5 electron volts. When dipolar molecules such as water or the polar side chains of proteins are exposed to the rapidly alternating electromagnetic fields, they oscillate through 1800 at the rate of 2.45 billion cycles per second. The molecular movement or kinetics so induced results in the generation of instantaneous heat that is proportional to the energy flux and continues until radiation ceases. Molecules other than water and the polar side chains of proteins including molecules with an uneven distribution of electrical charge such as inorganic material and copper oxides may also be made to oscillate in the electromagnetic field generated by MWs.

Pathos – a tragedy for conventional tissue processing

(Ref. PR-006)

Pathology In Practice
Sue Wollington
Read abstract…
Tissue processing changed little over the course of the last century until the introduction of microwave equipment into histopathology. Now, the technology that revolutionised work in the kitchen appears set to do the same in one of the oldest disciplines in pathology, as Sue Wollington explains.

A Novel fixative improves opportunities of nucleic acids and proteomic analysis in human archive’s tissues

(Ref. PR-007)

Diagnostic Molecular Pathology
Giorgio Stanta et al.
Read abstract…
All tissues from biopsy or surgery origin are fixed and paraffin embedded as a routine procedure in the hospital departments of pathology. The traditional method of tissue preservation is the fixation in formalin, followed by paraffin embedding. In this way tissue’s integrity is ensured also for future analyses, because there is no further chemical degradation of nucleic acids and proteins in tissues embedded in paraffin. After few sections for the histopathological examination the tissues are stored for decades in the hospital archives. Even if formalin fixation compromises the quality and integrity of nucleic acids, it has already been demonstrated that it is possible to recover and analyze DNA and RNA from these archive’s tissues, even of autopsy origin. Protein analysis is on the contrary completely blocked, due to the fact that formalin fixation creates covalent links between proteins and the only way to study protein expression is immunohistochemistry. In this study we present our results concerning the use of a new formalin free fixative, the FineFIX. After extraction of nucleic acids, PCR and RT-PCR analyses were performed in DNA and RNA respectively. For DNA analysis it was possible to obtain amplicons of 2400 bps, while in formalin-fixed samples the maximum length achieved was less than 400 bps. RT-PCR analysis show that it was possible to study RNA fragments of 600 bps from FineFIX fixed tissues, against a maximum length of about 150 bps achieved by formalin-fixed tissues. These tissues were analyzed also by Western Blot analysis, showing that the proteins obtained from FineFIX treated samples are amenable and comparable in quality with those obtained from fresh frozen tissues. Protein extracts from FineFix treated tissues were also compared with fresh tissues’ones by two dimensional electrophoresis, demonstrating that the protein pattern were well comparable for number and distribution of the spots.

Microwave Processing and Ethanol-Based Fixation in Forensic Pathology

(Ref. PR-008)

The American Journal of Forensic Medicine and Pathology
Antonio Iesurum et al.
Read abstract…
An ethanol-based fixative (FineFIX) has been used, together with rapid microwave-stimulated processing, in postmortem material, resulting in a rapid fixation and processing of the tissues with morphology, histochemical stains, and immunocytochemistry comparable to formalin-fixed material. Furthermore, this alternative fixation gives better DNA recovery in higher amounts if compared with DNA extracted from formalin-fixed tissue, particularly advantageous in forensic pathology.

Microwave Enhancement of CISH for HER2 Oncogene

(Ref. PR-009)

Applied Immunohistochemistry & Molecular Morphology
AnthonyS.-Y.Leong et al.
Read abstract…
We describe a modification to the prescribed procedure for the Zymed Spot-Light HER2 chromogenic in situ hybridization kit (84-0146, Zymed Laboratories, San Francisco,CA) by substituting the heat pretreatment step with MW irradiation in citrate buffer 10mmol/Lat pH 6.0 at 120°C for 10 minutes and repeating the procedure after enzyme digestion with time and temperature controlled in the MegaT/ T oven (Milestone s.r.l., Sorisole, Italy). The subsequent procedure leading up to hybridized was as per manufacturer’s instructions. Invasive breast carcinoma previously scoredby immunohistochemistry for HER2, comprising 18 cases of 3+, 18 cases of 2+, and 12 cases of 1+, were examined by chromogenic in situ hybridization using this modified procedure, with a parallel set of cases examined by the prescribed Zymed method. The introduction of the‘‘MW retrieval’’steps resulted in consistently a greater number of hybridization signals in amplified tumor cells with benign epithelial cells and lymphocytes displaying 2 clear dots compared with the weaker and less consistent signals obtained with the standard procedure. MW exposed sections showed larger numbers of large and small clusters that often allowed identificationof amplified tumors without having to count single dots with crisp staining and absence of background precipitation.

Definitive histologic diagnosis on prostate biopsies in 3 hours

(Ref. PR-010)

Department of Pathology. Vita-Salute San Raffaele University, Milan –Italy
L. Nava et al.
Read abstract…
Microwave-based devices allow ultra-rapid histoprocessing of bioptic specimens, surgical tissue blocks and whole organs. The aim of this study was to prospectively evaluate the efficacy and reproducibility of quickly processing prostate core needle biopsies with RHS1, an automated microwave vacuum processor.

A Microwave Tissue Processor Trial in a Routine Hospital Anatomical Pathology Laboratory

(Ref. PR-011)

Anatomical Pathology Laboratory, Canterbury Health Laboratories, Christchurch, New Zeland
Kevin Alderton et al.
Read abstract…
The Anatomical Pathology Laboratory at Canterbury Health Laboratories was given the opportunity to trial and evaluate the Milestone Histos 5 Microwave Tissue Processor (Fig. 1). In this evaluation over three months, a variety of tissues were sampled and processed using both conventional and microwave processing. The resulting tissue blocks were sectioned and the slides were stained using Haematoxylin and Eosin (H&E) as per normal laboratory operating procedures. A small number of immunohistochemistry and special stains were also performed. Stained slides were evaluated by a panel of seven pathologists. A grading system for the H&E slides was established and all of these slides were scored against this grading system.

Vision quest – fresh look at AP automation

(Ref. PR-012)

Cap Today
Karen Titus
Read abstract…
Complete voice dictation and a same-day biopsy service are part of the makeover of the AP laboratory at Holland (Mich.) Hospital. “We have what we call the clean-table club- you clean out all the cases before you go home” says Dr. Edward Fody.

Clinical Electron Microscopy

(Ref. PR-013)

Imaging & Microscopy
Dr. Josef A. Schröder
Read abstract…
The high resolution and sensitivity of electron microscopy is a valuable ancillary tool or gold standard in pathological diagnosis. The conventional sample turnaround time for processing in the lab can be significantly reduced from days to hours by the microwave technology.

Impact of Microwave-Assisted Sample Preparation in Diagnostic Electron Microscopy Today

(Ref. PR-014)

Central Laboratory of Electron Microscopy, Dept. of Pathology, University Hospital Regensburg, Germany
Josef A Schroeder et al.
Read abstract…
The primary basis for pathologic tissue diagnosis is the morphological analysis: a pathologist assesses by light microscopy cell and matrix appearances including architecture on an H&Estained section of tissue embedded in paraffin wax, in context of gross findings and clinical data, to render a diagnosis. Additional stains and techniques, like immunohistochemistry (IHC), flow cytometry, cytogenetics and “molecular” techniques (gene rearrangement analysis, fluorescence in situ hybridization and polymerase chain reaction (PCR) analysis) provide additional information to refine the understanding of disease and diagnosis. Electron microscopic (EM) examination is a method of extending morphologic analysis to the ultrastructural level providing information not discernible by the other methods, e.g. on the basis of the antigens expressed by a neoplasm. The advent and continual development and automation of the ancillary techniques, especially in IHC and now in molecular biology, resulted in reduced importance of the EM in pathology since the early 1980s. Also a number of limitations of the EM (e.g. sample error, need for adequate fixation, expensive and sophisticated instrumentation, long turnaround time, high level of staff skills and interpretation expertise) comes along. Today, EM-diagnostic expertise is generally available only in larger laboratories or centres with specific interest in EM.

Microwave histoprocessing versus conventional histoprocessing

(Ref. PR-015)

Indian Journal of Pathology and Microbiology
Alka Mary Mathai et al.
Read abstract…
The aim of the study is to compare the histologic quality of the microwave histoprocessing with that of conventional method and to determine its positive impact on turnaround times and reduction of costs of tissue processing. One hundred and eighty-five paired tissue sections from different organs were taken. Each tissue sections were of size of 15 mm x 10 mm x 3 mm and divided into two; one set as experimental group and the other as control group. The tissues in the experimental group were further divided into six groups and processed by vacuum-microwave method according to six protocols from I to VI. Other tissues in the control group were processed by the conventional method and compared. Overall, the quality of microscopic tissue from both the methods was identical. Microwave processing shortened the time of processing without compromising the overall quality of the histologic section and was cost-effective.

Pathos processing: the Liverpool experience

(Ref. PR-016)

Pathology In Practice
Emma Colgan
Read abstract…
Conventional tissue processing in histopathology can be a time-consuming exercise. However, use of the very latest technology is having a major impact on working patterns in histopathology, as Emma Colgan explains.

Revolutionary New Walk in Skin clinic

(Ref. PR-017)

Quest Diagnostics
Rob Ford
Read abstract…
Quest Diagnostics have teamed up with the Cadogan Clinic on London’s Sloane Street and are opening a brand new Walk in Skin Clinic. The general public will simply be able to walk into the Clinic in the morning and receive the results of their biopsy in the afternoon.

Microwave Processing in the Histology Lab

(Ref. PR-018)

Johns Hopkins Institute, New York, USA
Deborah Duckworth
Read abstract…
In almost every lab across the country, efforts to improve turnaround times, provide better service to patients and maintain a safe environment for staff are at the forefront. We are no different, if anything we have higher expectations placed upon us as the premier hospital in the nation. It is because of this fact that an effort to change “how we’ve always done it” has come with mixed reviews. Routinely, the processing of a specimen in Surgical Pathology begins in accessioning, then moves to grossing for sectioning and is placed into fixative, normally 10% Neutral Buffered Formalin. The tissue cassettes are then picked up by a Histotech at the end of the day, loaded onto a standard tissue processor on a 12 hour overnight processing schedule. The goal of processing is to remove all of the water from the tissue through a series of graded alcohols and xylenes; and then infiltrate the tissue with paraffin wax. This will allow the tissue to then be embedded, sectioned and placed on a slide for staining and pathologists’ review. This process has not changed in almost 100 years, this is histology. Recently, as in the past 30 years, microwave technology has been explored as an alternative to routine processing. Microwaves (electromagnetic waves) are short pulses of energy that allow the solution that the tissue cassettes are submersed in to be heated evenly and are able to maintain that temperature over time. The overall benefit from this heat element alone is that the fixation and dehydration of the tissue occurs in about one third of the time. In essence, a routine specimen such as a radical prostatectomy, grossed at a thickness of 3mm, can be processed by our microwave processor in about 3 hours 45 minutes in comparison to a standard 12 hour processing schedule.

Virtual slide telepathology enables an innovative telehealth rapid breast care clinic

(Ref. PR-019)

Human Pathology
Ana Maria López et al.
Read abstract…
An innovative telemedicine-enabled rapid breast care service is described that bundles telemammography, telepathology, and teleoncology services into a single day process. The service is called the UltraClinics Process. Because the core services are at 4 different physical locations, a challenge has been to obtain stat second opinion readouts on newly diagnosed breast cancer cases. To provide same day quality assurance rereview of breast surgical pathology cases, a DMetrix DX-40 ultrarapid virtual slide scanner (DMetrix Inc, Tucson, AZ) was installed at the participating laboratory. Glass slides of breast cancer and breast hyperplasia cases were scanned the same day the slides were produced by the University Physicians Healthcare Hospital histology laboratory. Virtual slide telepathology was used for stat quality assurance readouts at University Medical Center, 6 miles away. There was complete concurrence with the primary diagnosis in 139 (90.3%) of cases. There were 4 (2.3%) major discrepancies, which would have resulted in a different therapy and 3 (1.9%) minor discrepancies. Three cases (1.9%) were deferred for immunohistochemistry. In 2 cases (1.3%), the case was deferred for examination of the glass slides by the reviewing pathologists at University Medical Center. We conclude that the virtual slide telepathology quality assurance program found a small number of significant diagnostic discrepancies. The virtual slide telepathology program service increased the job satisfaction of subspecialty pathologists without special training in breast pathology, assigned to cover the general surgical pathology service at a small satellite university hospital.

Histoprocesorul cu Microunde faciliteaza diagnosticarea rapida a cancerului colorectal

(Ref. PR-020)

Spitalul urgenta “dr.Agrippa Ionescu” Bucuresti, Romania
Florin Obrocea and Maria Sajin
Read abstract…
The Histoprocessor with Microwave facilitates the rapid diagnosis of colorectal cancer

An Initial Experience with Rapid Microwave Processing in the One-Stop Breast Clinic

(Ref. PR-021)

World Journal of Surgery
Edward Parkin et al.
Read abstract…
Background: Rapid microwave processing allows core biopsy results to be obtained within a 3- to 4-h time period. This study was designed to compare the accuracy and reporting time of microwave-processed breast biopsies with samples processed using traditional methods. Methods: Concordance of the preoperative biopsy report with postoperative histology for tumor type, grade, and detection of lymphovascular invasion was recorded for both techniques. Also reviewed were the time taken between day of biopsy and day of reporting, waiting time between biopsy and surgery, and number of preoperative outpatient appointments. Results: In the microwave-processed group (MG; n = 43), there was a 92% concordance rate between preoperative biopsy and postoperative histology for tumor type. In the traditional group (TG; n = 43), it was 80% (P > 0.05). For tumor grade, there was a concordance rate of 64% in MG and 93% in TG (P > 0.05). For detection of lymphovascular invasion, there was agreement in 88% of cases in MG and 67% in TG (P > 0.05). Twenty-five patients from MG were informed of their diagnosis on the day of biopsy. There was no difference in waiting time from biopsy to surgery or number of preoperative outpatient appointments between MG and TG (P > 0.05). Conclusions: Microwave processing allows safe and accurate immediate histological reporting. As a result, surgical management can be planned at the initial outpatient consultation.

Integration of microwave technology to reduce fixation and processing time of robotic prostatectomy

(Ref. PR-022)

Department of Pathology, Henry Ford Health System, Detroit, Michigan, USA
R. Zarbo et al.
Read abstract…
Background: Using the Lean tool of value stream mapping, we identified the preanalytic phase of processes from specimen receipt to tissue processing as the major source of time delay and non-value added effort in the value stream of whole mount prostatectomy evaluation. Design: We targeted intact gland formalin fixation and tissue processing of whole mount (WM) sections. The initial condition consisted of 50-100ml formalin needle injection of intact robotic prostatectomies using a fine needle. Intact glands were then fixed overnight in 32 oz. formalin for avg. 15.8 hours. Glands were entirely sectioned at 4mm thickness and WM sections were fixed for 9 hrs. in macro-cassettes (LPC1000, Cancer Diagnostics Inc) and processed for 16.5 hrs. in a VIP 300E processor (Sakura Finetek USA, Inc, Torrance, CA). The redesigned condition retained formalin injection of glands that were then fixed intact in 32 oz. of formalin for 30 min. followed by microwave fixation in 32 oz. of formalin in a microwave processor (Model EBS42850, EBSciences, East Granby, CT) at 450W for 6 min. at 50C°. Glands over 50 grams were held for 15 additional min. in formalin at room temperature then microwaved again for 3.5 min. WM sections were then fixed in macrocassettes for an additional 2 hrs. at room temperature. Total fixation time in the redesigned process was 2.53 hrs. WM sections were processed in 7 hrs. in macro-cassettes in a Pathos DELTA microwave processor (Milestone Medical Technologies, Inc, Kalamazoo, MI/Sirasole, Italy). Baselines were from 20 robotic prostatectomies. New conditions were tested on 5 autopsy and 10 clinical glands.The latter were parallel tested with WM slices from each gland in the VIP and Pathos DELTA processors. All microscopic sections were assessed by one GU pathologist. Results: With process redesign fixation time was reduced by 90% from 24.8 to 2.53 hrs. Integrating microwave technology in tissue processing reduced processor time by 58% from 16.5 to 7 hrs. Overall, 32 hrs. of non-value added time waste was removed from the front end processes resulting in a 77% reduction from 41.3 to 9.5 hrs. Pathologic examination showed no significant variations in quality related to H&E staining, homogeneity, crust/edge effect, completeness of margins, nuclear preservation, autolysis or thermal artifact. Conclusions: Lean process redesign targeting intact prostate gland fixation and integration of microwave technology in radical prostatectomy processing can be of great benefit in addressing total timeliness of pathologic reporting.

Optimization of fixation and processing of biopsy

(Ref. PR-023)

Department of Pathology, Henry Ford Health System, Detroit, Michigan, USA
RJ Zarbo et al.
Read abstract…
Background: Consistency of nuclear and cytologic detail in prostate needle biopsy specimens is a critical aspect of histopathologic diagnosis. The preanalytic parameters of formalin fixation from time of surgical procedure are largely uncontrolled and inadequate fixation may contribute to less than optimal histology when using same-day rapid tissue processing. To accommodate this unstandardized variable by defaulting to overnight fixation of Biopty gun prostate needle biopsies negates the advantage of rapid cycle processing. Design: We performed Biopty Gun (Bard Peripheral Vascular, Inc. Tempe, AZ) needle biopsies of fresh clinical prostatectomy specimens to test 18 pathway variations of tissue fixation and processing. Needle (18G) cores obtained from the posterior aspect of glands (8 per side) were 1 mm or less diameter and averaged 15-18 mm length. To control for variation in fixation time and mimic air-drying, cores were placed on saline soaked gauze initially before testing combinations of no fixation other than on processors, timed tissue fixation at room temperature, desktop microwave formalin fixation for 3.5 minutes (Model EBS42850, Energy Beam Sciences, East Granby, CT), controlled heated fixation for 30 minutes at 37°, 45° and 50°C (FixMate, Milestone Medical Technologies, Inc, Kalamazoo, MI/Sirasole, Italy) and processing on two microwave enhanced instruments, Tissue-Tek Xpress (Sakura Finetek USA, Inc, Torrance, CA) and Pathos DELTA (Milestone Medical Technologies). Quality assessments were made of ease of embedding and microtome sectioning and histopathologic variables of hematoxylin and eosin staining intensity, homogeneity, nuclear preservation, nucleolar prominence, autolysis or thermal artifact. Microscopic sections were evaluated by one GU pathologist. Microscopic quality was scored on a 10-part scale. Results: With the exception of the FixMate heated and timed fixation tests at 37°C and 45°C, all combinations gave inconsistent and spotty unacceptable, suboptimal to good microscopic preparations no matter the variable manipulated. The higher 50°C test of heated formalin fixation resulted in an artifact of nuclear chromatin that was dark and smudgy. The histologic quality at 37°C was judged slightly superior to 45°C. No significant difference in quality of tissue microtomy or histologic preparation was noted for either microwave tissue processor. Conclusions: Enhanced consistency in the quality of histologic preparations using rapid microwave processors is obtained when prostate needle biopsy fixation is standardized with controlled time and temperature of fixation.

One-day core needle biopsy in a breast clinic: 4 years experience

(Ref. PR-024)

Breast Cancer Research and Treatment
Joris P. Bulte et al.
Read abstract…
Many attempts have been made to combine the high diagnostic accuracy and conclusive rate of core needle biopsy (CNB) with the speed of fine needle aspiration cytology in evaluation of solid breast lesions. Multiple hybrid techniques have been developed to achieve this. We describe a cohort of patients for whom we used a relatively new, accelerated method of CNB processing, allowing for a definitive diagnosis the same day. All patients visiting the Radboud University Nijmegen Medical Centre breast clinic during a 4-year period were reviewed to identify all CNBs in this period performed in a same-day diagnosis track. CNB result was compared to post-operative pathology reports when available, and to follow-up when patients were not surgically treated. 1,060 patients underwent CNB of 1,383 lesions, 898 of which in a same-day diagnosis track with a sensitivity of 96.9 % and a specificity of 99.4 %. The inconclusive rate was 9.2 %. For a same-day diagnosis for solid breast lesions, we could give a conclusive diagnosis with accelerated CNB processing in 65 % of our patients requiring CNB. This technique can be used reliably in a same-day diagnosis breast clinic with a very high sensitivity, specificity, and conclusive rate.

Validation of Histology Tissue Processing and Stain Quality of Logos Rapid?Cycle Microwave Processor in Lean Continuous Flow Operations

(Ref. PR-025)

Department of Pathology, Henry Ford Health System, Detroit, Michigan, USA
Richard J Zarbo et al.
Read abstract…
Pathology report timeliness can be enhanced by technology that reduces time waste in histology. The largest delay is overnight fixation and prolonged processor times. These throughput bottlenecks can be targeted by integrating tissue processors capable of rapid-cycle times especially when aligned with continuous flow Lean work design.

Evaluation of Xylene-free Tissue Processing on the LOGOS all-in-one Hybrid Tissue Processor

(Ref. PR-026)

Pathology Department , Aberdeen Royal Infirmary, Aberdeen, Scotland
Lynne Doverty and Linda Davidson
Read abstract…
The Pathology department at Aberdeen Royal Infirmary was in a position to replace one of their tissue processors. As part of this process, we considered same-day processing and also the Health & Safety issues concerning xylene. We investigated several of the processors on the market, Milestone LOGOS tissue processor fulfilled our requirements: – Xylene-free processing – Microwave technology for same-day processing Two members of staff were invited to a demonstration in the Milestone factory near Milan and were impressed by the LOGOS, therefore an on-site demonstration was arranged. The LOGOS was on trial in the department for 2 weeks over which time various tissue blocks were processed on 3 different protocols. H&E, Special stains and Immunohistochemistry were carried out on the sections.

Comparación entre el Histoprocesamiento Convencional y en Microondas KOS por medio de Tinción de Rutina, Histoquímica e Inmunohistoquímica en Maxilares de ratones Mus Musculus

(Ref. PR-027)

Alumna Pregrado de Tecnología Médica, Mención Morfofisiopatología y Citodiagnóstico, Universidad de Valparaíso, Chile
Salinas, D. et al.
Read abstract…
RESUMEN: En la actualidad es necesario implementar en el Laboratorio de Anatomía Patológica, nuevas tecnologías que ayuden a disminuir el tiempo y toxicidad del histoprocesamiento y que aseguren la mantención de la calidad histológica e integridad del operador. Una herramienta útil es el microondas, con la introducción del modelo KOS, modelo CAT. 67051 de MILESTONE MEDICAL TECHNOLOGIES. Se seleccionaron maxilares de 14 ratones Mus Musculus. Fueron seccionados y sometidos a un histoprocesamiento, que involucró el procesador automático convencional y el microondas KOS, respectivamente. Se confeccionaron matrices de valoración, para calificar el desempeño de cada histoprocesamiento, a través de criterios de corte y coloración de tinciones de rutina (H-E, Tricrómico de Masson), histoquímica (PAS) e Inmunohistoquímica (Desmina, PCK y ?-actina). La observación y evaluación de las láminas se realizó por tres observadores, ciegos para el estudio. Se utilizó el Test de Wilcoxon (p < 0.05) para analizar los datos. No existieron diferencias estadísticamente significativas, en el resultado final, al comparar ambos histoprocesamientos. El microondas de laboratorio KOS surge como una herramienta útil y que se puede emplear con seguridad en el Laboratorio de Anatomía Patológica.

Use of microwave in diagnostic pathology

(Ref. PR-028)

Journal of Cancer Research and Therapeutics
Basavaradhya Sahukar Shruthi et al.
Read abstract…
Conventional tissue fixation and processing is as old as 100 years and still remains the gold standard against which all new technologies and methods need to be assessed. Tissue processing is one of the important steps for obtaining good thin sections without artifacts. Though conventional tissue-processing methods are most commonly followed, they are well-known as very laborious and tedious procedures. Microwaves a form of electromagnetic wave-induced heat, when applied in histotechnology, reproducibly yields histolologic material of similar or superior quality to that provided by conventional processing methods, making it more popular in the recent years. A laboratory microwave offers features like maximum output of 2000-3000 watts, an in-built source of adjustable temperature probe, facility for ventilation of hazardous fumes, but is expensive. Considering the usefulness of microwave in histotechnology, i.e., reducing the time required for the diagnosis, replacing the conventional equipments of laboratories by microwave-guided ones is a remarkable and an acceptable change.

MICROWAVE PROCESSING FOR LabPlus Trial of KOS

(Ref. PR-029)

Anatomical Pathology, LabPlus, Auckland, New Zeland
Linda Graham et al.
Read abstract…
LabPlus EM trial of the KOS microwave commenced in March 2012 and trials continued for another 6 weeks after the initial training. The most time consuming aspect was the loading & unloading of tissue into the processing baskets. Hopefully this is something that would speed up with more practice. At this point in time only the small tissues have been viewed on the TEM. Only a few LR white processed blocks had curing & sectioning problems. Preliminary results show improved infiltration (& consequently improved sections) in larger tissues.

Microwave-Assisted Processing and Embedding for Transmission Electron Microscopy

(Ref. PR-030)

Electron Microscopy: Methods and Protocols, Methods in Molecular Biology
Paul Webster
Read abstract…
Microwave processors can provide a means of rapid processing and resin embedding for biological specimens that are to be sectioned and examined by transmission electron microscopy. This chapter describes a microwave-assisted protocol for processing, dehydrating and embedding biological material, taking them from living specimens to blocks embedded in sectionable resin in 4 h or less.

Rapid processing of bone marrow biopsies (BMB’s) with excellent morphology, immunohistochemistry and DNA quality

(Ref. PR-031)

Department of Pathology, Radboud University Medical Centre, Nijmegen, the Netherlands
I. de Laak–de Vries et al.
Read abstract…
BMB is an essential part of the diagnosis in patients with haematological diseases. In line with other oncological fast diagnostic tracks we optimized the processing of BMB’s to achieve reporting the following day.

How we do: optimizing bone marrow biopsy logistics for sign-out within 2 days

(Ref. PR-032)

Journal of Hematopathology
I. de Laak–de Vries et al.
Read abstract…
Since the introduction of fast diagnostic tracks in many areas of oncology, the traditional processing of bone marrow biopsies (BMB), requiring either resin embedding or lengthy fixation and decalcification, is due to an upgrade. Thanks to a growing number of new commercially available tissue processors, microwave-enhanced processing is becoming a standard tool in the pathology laboratory, allowing rapid fixation and decalcification of BMB with preserved morphology and antigens. In this short report, we describe the use of a commercially available EDTA-based decalcification fluid (USEDECALC, Medite, Orlando, USA) in combination with the LOGOS J (Milestone, Bergamo, Italy), a closed microwave-enhanced tissue processor, for overnight fixation, decalcification, and paraffin impregnation of the BMB. This allows next-day reporting without impaired morphology or immunohistochemistry, and even improved DNA quality of the BMB.

Breast Biomarkers Can Be Reliably Tested on Tissue Fixed and Processed by Same Day Automated Microwave-Assisted Method

(Ref. PR-033)

Sunnybrook Health Sciences Centre, University of Toronto, Ontario, Canada
Elzabieta Slodkowska et al.
Read abstract…
BACKGROUND – Recommendations to validate any modification in breast cancer biomarker testing have been endorsed by CAP and other regulatory bodies to ensure accurate testing – Accordingly, any proposed modification has to be tested against a previously validated technique. For modifications in preanalytical variables this entails creating a validation set of paired samples, consisting of a combination of positive (P), low positive (LP, < 10%) and negative (N) cases that have been prepared under the same conditions and differing only with respect to the modified variable - Microwave-assisted rapid tissue fixation and processing (MWARTFP) significantly reduces tissue processing time

Brief fixation enables same-day breast cancer diagnosis with reliable assessment of hormone receptors, E-cadherin and HER2/Neu

(Ref. PR-034)

Journal of Clinical Pathology
Altuna Halilovic et al.
Read abstract…
Aims: Preoperative core needle biopsy (CNB) is commonly used to confirm the diagnosis of breast cancer. For treatment purposes and for determining histological type, especially in case of neoadjuvant therapy, oestrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) status and E-cadherin assessments are crucial. Considering the increasing demand for same-day diagnosis of breast lesions, an accelerated method of CNB processing was developed, in which the tissue fixation time is radically reduced. Methods: To determine whether short fixation time frustrates assessment of ER, PR and E-cadherin immunohistochemistry (IHC) and HER2 fluorescence in situ hybridisation (FISH), 69 consecutive patients with 70 invasive breast carcinomas were included through the same-day diagnostics programme of breast lesions of the Radboud university medical center and the hospital Pantein. IHC for ER, PR and E-cadherin and HER2 FISH were compared between CNBs fixed for approximately 60-90 min and traditionally fixed resection specimens. Results: Overall agreement between CNBs and resection specimens was 98.6% for ER (p<0.001; ?=0.93), 90.0% for PR (p<0.001; ?=0.75), 100% for E-cadherin (p<0001; ?=1.00) and 98.6% (p<0.001; ?=0.94) for HER2 FISH. Positive and negative predictive values were respectively 98.4% and 100% for ER, 95.9% and 76.2% for PR, 100% and 100% for E-cadherin and 90% and 100% for HER2 FISH. Conclusions: Hormone receptors and E-cadherin IHC and HER2 FISH are highly comparable between briefly fixed CNBs and the corresponding traditionally fixed resection specimens, and can therefore reliably be used in the daily clinical practice of same-day diagnostics of breast cancer.

Whole Mount Histological Preparations for Prostate and Kidney

(Ref. PR-035)

UT Southwestern Medical Center, Texas, USA
Archana Mainali et al.
Read abstract…
Preparing whole mounted Histology preparations of diagnostic quality provides a significantly superior Histopathological registration for surgical specimens that have undergone in vivo MRI. The whole mounted slides offer excellent Pathology correlation with MRI imaging derived, 3D printed patient specific whole mount molds for prostate and kidney surgical specimens. An important requirement for MRI Pathology correlation is adequate image registration. Histopathologic analysis using standard tissue based processes where the prostate and kidney are sectioned in multiple routine tissue blocks results in challenges of correlation between the in vivo MRI findings and the reference Histopathological standard. Patient specific 3D printed mold was created utilizing the in vivo MRI data and utilized to prepare slabs of tissue for processing. Specimen orientations were marked with tissue color and grossed into mega cassettes with adequate slot perforations for good reagent exchange. Processing schedule was developed on the Milestone LOGOS microwave processor for specimens of a standardized tissue thickness of 5 mm. The protocol was optimized to produce diagnostic quality histologic preparations in addition to good tissue preservation for immuno-histochemical and molecular studies. A standard specimen orientation was mainted during embedding to ensure optimal visualization of lesion tissue. Standard microtomes were retrofitted with chucks to accomodate mega cassettes. High quality tissue whole sections were obtained utilizing specialized microtome blades on large charged slides. The slides were placed on rack adapters and H&E stained on routine histology stainers. Specific tissue components would be marked by a Pathologist for further studies including special stains, IHC and molecular that would assist technologist prepare sections or tissue on regular slides. In the current design radiologist and pathologist work independently for diagnostic medicine. Whole mounted histological preparations on 3D printed patient specific MRI offers a unique opportunity for the two disciplines to collaborate. In this setting pre-operative multi-parametric MRI data when correlated with whole mounted Histo-Pathological findings can provide valuable insights into MRI detected suspicious lesions by assissting in characterizing the tumor and assigning a Gleason score before surgical intervention.

Microwave Tissue Dehydration. A Fast and Reliable Tissue-Processing Method in Pathology

(Ref. PR-036)

Analytical and quantitative cytology and histology
Yanwei Li et al. 
Read abstract…
OBJECTIVE: To compare the histologic quality of tissues processed with rapid microwave tissue dehydration and traditional tissue dehydration. STUDY DESIGN: A total of 200 paired tissues from 15 organs were taken and diveded into 2 groups. One group was processed with rapid microwave tissue dehydration, and the other was processed with traditional tissue dehydration. Hematoxylin and eosin (H&E), immunohistochemical (IHC), and in situ hybridization (ISH) staining were conducted, and staining qualities were compared carefully. Evaluation of blocks, H&E stain, IHC stain, and ISH were carried out. RESULTS: the quality of blocks and H&E stain showed no distinction. The IHC signal-positive location, percentage of positive cells, grades of intensity, and the staining background showed a very high concordance between the 2 groups. The ISH results show no distinction from each other. Moreover, microwave tissue dehydration is time-saving. CONCLUSION: As a rapid and reliable tissue processing method, microwave tissue dehydration may potentially be useful when rapid pathological diagnosis is needed.

A New Fully Automated Processing and Embedding Technique for Cytological Cell Blocks

(Ref. PR-037)

Biomedical – Journal of Science & Technical Research
Campora Michela et al.
Read abstract…
Cell blocks are a valuable, widely demonstrated tool for obtaining paraffin-embedded cell sections on which to perform standard staining procedures and, when appropriate, immunohistochemistry or other ancillary tests. We describe an innovative application of a fully automated inclusion and embedding procedure, routinely used for small biopsies, to create cell blocks thereby simplifying methodology and improving quality for cytological diagnosis.

Is it possible to reduce processing time for mamma core biopsies by using Pathos Delta 1 mm program

(Ref. PR-038)

Department of Pathology, Herlev and Gentofte Hospital, University of Copenhagen, Denmark
Lone Bojesen et al.
Read abstract…
In our laboratory we process mamma core biopsies on Tissue Tek VIP 5 (VIP) overnight for 13 hours, after a minimum of 4 hours fixation in 4% neutrally buffered formaldehyde (NFB). This is to ensure the most optimally processed tissue for downstream analysis. This means that mamma core biopsies fixed after 1 pm is delayed 24 hours. Aim: to test a shorter program for processing 1 mm core biopsies (1 hour and 27 minutes) after 30-60 minutes fixation in NBF on Pathos Delta, and compare all analysis routinely performed on mamma core biopsies after the standard VIP processing.

Accelerated Tissue Processing With Minimal Formalin Fixation Time for 9-Gauge Vacuum-Assisted Breast Biopsy Specimens

(Ref. PR-039)

American Journal of Clinical Pathology
Joris P. Bulte et al.
Read abstract…
Objectives: Vacuum-assisted biopsy (VAB) of the breast seems unsuitable for rapid processing due to large size. We tested microwave-based acceleration. Methods: As a proof-of-principle study, 9-gauge VAB specimens were taken from eight mastectomy specimens. Forty-two biopsy specimens were processed. Quality of H&E was evaluated in 84 slides, and estrogen receptor (ER), progesterone receptor (PR), E-cadherin, and human epidermal growth factor receptor 2 (HER2) stains were evaluated in six slides. Preoperative biopsy specimens were used as a control. Results: Diagnostic quality of H&E slides was good in 87%, reasonable in 12%, and low in 1%. Quality of E-cadherin was good in 75% and reasonable in 25%. Quality of ER was good in 83% and reasonable in 17%. PR and both HER2 immunohistochemistry and fluorescence in situ hybridization were good in all slides. Quality of experimental slides was similar to control slides. Conclusions: Nine-gauge VAB specimens can be processed within 4 hours. Slides are suitable for all routine pathologic stains. This enables a same-day diagnosis.

The influence of processing platform on morphology, immunohistochemistry and DNA quality

(Ref. PR-040)

Department of Pathology, Herlev and Gentofte Hospital, University of Copenhagen, Denmark
Lone Bojesen et al.
Read abstract…
In our laboratory we routinely process intestine biopsies and skin samples on the Tissue Tek Xpress for 1 to 2 hours, but in a previous project done on intestine biopsies, we found that especially the DNA analysis was negatively affected by this. We therefore wanted to test another rapid processing platform Pathos Delta and its program for 3 mm samples. Aim: to test the influence of processing platforms on morphology, immunohistochemistry and DNA quality.

Step-by-step preparation of mouse eye sections for routine histology, immunofluorescence, and RNA in situ hybridization multiplexing

(Ref. PR-041)

Cell Press
Ji Pang et al.
Read abstract…
It can be challenging to maintain tissue integrity using established histology protocols. Here, we describe a protocol composed of Hartman’s fixation, window technique, microwave-based tissue processing, optimized depigmentation, and antigen retrieval pretreatment. This is followed by the ViewRNA single-molecule fluorescence in situ hybridization and immunofluorescence techniques to optimize routine histological staining and molecular histology multiplexing assays. Our protocol is highly reproducible in any laboratory and may decrease animal usage and lab resource expenditure. For complete details on the use and execution of this protocol, please refer to Pang et al. (2021).

Reagents

Ultra-Rapid Microwave/Variable Pressure-Induced Histoprocessing: Description of a New Tissue Processor

(Ref. RE-001)

The Journal of histotechnology
F.Visinoni et al.
Read abstract…
We describe a new method of ultra-rapid histoprocessing that reduces the processing times for needle and endoscopic biopsies to 30 min and that of other surgical biopsy tissue blocks of up to 4 mm thick to 120 min. The MicroMED U R M Histoprocessor, which combines microwave irradiation with precise computer control of power, time, temperature, vacuum, and pressure, when used with a 1-step dehydrating/clearing reagent, consistently produces rapidly processed tissues with optimal cytomorphology and improved tissue sectioning properties. Staining properties are excellent with no deleterious effects on routine staining, histochemistry, or immunohistochemistry. This new processing technique represents a major change from conventional tissue processing and eliminates the use of hazardous reagents such as xylene. The ease of application and speed of this technique can significantly reduce turnaround times in diagnostic laboratories.

A formalin-free fixative, that provides superior preservation for molecular techniques

(Ref. RE-002)

University Hospital of Birmingham, UK
GM Reynolds et al.
Read abstract…
The aim of fixation is the preservation of cells and tissue constituents in as life like a form as possible. Despite the search for many years, using numerous combinations of fixing agents and additives, the ‘ideal’ fixative has alluded histologists. Diagnostically, the most important feature has always been tissue architecture. More recently, over the last three decades the preservation of proteins within tissues has become more vital, with the evolution of immunohistochemical techniques. It is therefore not surprising that the fixative of choice for routine histological diagnosis, has remained formaldehyde-based. These not only provide excellent morphological preservation, but also protein preservation. The fact that proteins, or more specifically, the antigenic sites that are required to be demonstrated by immunohistochemical techniques become ‘masked’ due to chemical interactions provided a small set back. Formalin fixation is a reversible reaction, so with the evry increasing importance of antobodies as diagnostic tools ways to reverse the maskingof antigenic sites were developed. Now however, we are seeing a new development in routine laboratory practise, the need to perform essential molecular diagnostic and prognostic techniques. The feature required for the “ideal” fixative have now shifted. Formaldehyde-based fixatives are poor preservatives of DNA and RNA. Whilst it could be argued alcohol based fixatives are good for this, they result in poor morphology and to a certain degree tissue antigenicity which are still essential diagnostic criteria. In this study we evaluate a novel formalin-free fixative, FineFIX, which proposes to offer a combination of good morphological preservation along with preservation of tissues in such a way as to allow essential molecular profiling techniques.

A novel fixative improves opportunities of nucleic acids and proteomic analysis in human archive’s tissues

(Ref. RE-003)

Diagnostic Molecular Pathology
Giorgio Stanta et al.
Read abstract…
All tissues from biopsy or surgery origin are fixed and paraffin embedded as a routine procedure in the hospital departments of pathology. The traditional method of tissue preservation is the fixation in formalin, followed by paraffin embedding. In this way tissue’s integrity is ensured also for future analyses, because there is no further chemical degradation of nucleic acids and proteins in tissues embedded in paraffin. After few sections for the histopathological examination the tissues are stored for decades in the hospital archives. Even if formalin fixation compromises the quality and integrity of nucleic acids, it has already been demonstrated that it is possible to recover and analyze DNA and RNA from these archive’s tissues, even of autopsy origin. Protein analysis is on the contrary completely blocked, due to the fact that formalin fixation creates covalent links between proteins and the only way to study protein expression is immunohistochemistry. In this study we present our results concerning the use of a new formalin free fixative, the FineFIX. After extraction of nucleic acids, PCR and RT-PCR analyses were performed in DNA and RNA respectively. For DNA analysis it was possible to obtain amplicons of 2400 bps, while in formalin-fixed samples the maximum length achieved was less than 400 bps. RT-PCR analysis show that it was possible to study RNA fragments of 600 bs from FineFIX fixed tissues, against a maximum length of about 150 bs achieved by formalin-fixed tissues. These tissues were analyzed also by Western Blot analysis, showing that the proteins obtained from FineFIX treated samples are amenable and comparable in quality with those obtained from fresh frozen tissues. Protein extracts from FineFixr treated tissues were also compared with fresh tissues’ones by two dimensional electrophoresis, demonstrating that the protein pattern were well comparable for number and distribution of the spots.

Microwave processing and ethanol-based fixation in forensic pathology

(Ref. RE-004)

The American Journal of Forensic Medicine and Pathology
Antonio Iesurum et al.
Read abstract…
An ethanol-based fixative (FineFIX) has been used, together with rapid microwave-stimulated processing, in post mortem material, resulting in a rapid fixation and processing of the tissues with morphology, histochemical stains, and immunocytochemistry comparable to formalin-fixed material. Furthermore, this alternative fixation gives better DNA recovery in higher amounts if compared with DNA extracted from formalin-fixed tissue, particularly advantageous in forensic pathology.

Determination of FineFIX fixation on histologic quality and recovery of RNA from human prostate tissue

(Ref. RE-005)

National Cancer Institute, Bethesda
Benjamin Espina et al.
Read abstract…
Preserving tissue specimens essentially entails either fixation and embedding or snap-freezing. Traditionally, fixation and embedding protocols have been used to preserve tissue for histologic analysis without regard to biomolecular preservation. Snapfreezing has been the gold standard for bimolecular preservation but poorly preserves histologic characteristics. It would be ideal to develop a fixation and embedding protocol that would not only maintain excellent histology, but would preserve biomolecules to the same extent as snap-freezing. Examining prostate tissue, this study compares FineFix-fixed/paraffinembedded (FiPE) tissue with tissue that has been snapfrozen, formalin–fixed/paraffin-embedded (FFPE), and 70% ethanol-fixed/paraffin-embedded (EFPE) for histology and recovery of RNA. Tissue sections are H&E stained for histologic analysis and for RNA analysis, epithelial cells are microdissected, RNA purified, then reverse transcribed followed by polymerase chain reaction (PCR) using primers specific for 220 bp beta actin. The PCR products are run on an agarose gel. Histologic quality was comparable for FFPE, EFPE, and FiPE and were all superior to frozen. RNA recovery was comparable for frozen and FiPE and were superior to EFPE and FFPE. Tissue fixation using FineFix may be useful for performing gene expression analysis from microdissection of different cell populations showing subtle histologic differences.

Articular cartilage and synovial tissue

(Ref. RE-006)

BioSyntech Inc., Montréal, Canada
E. Rossomacha et al.
Read abstract…
Standard histoprocessing for hard tissue or sensitive soft tissue typically involves a lengthy manual process. Advances in microwave (MW) technology offer new opportunities to facilitate histoprocessing without compromising results. In 1998, a new one-step dehydration/clearing agent called JFC solution (Milestone, Italy) was developed specifically for use in MW histoprocessing. JFC solution consists of absolute ethanol, and isopropyl alcohol with long-chain hydrocarbons and has transformed histoproccesing into a 2-step process: JFC and paraffin, and effectively reduced processing time to between 30 minutes and 3 hrs (depending on size and nature of tissue). Here we compared the histological quality of rabbit femoral articular cartilage and patellar synovial tissue following histoprocessing using two different methods: a) routine manual and b) MW processing with JFC solution for dehydration and clearing in one step.

Assessment of different fixatives, fixation and tissue processing on morphology, antigenicity, and acid nucleic integrity from tumoral and non tumoral thyroid tissues

(Ref. RE-007)

Pasteur Hospital, CHU de Nice, France
Virginie Gavric-Tanga et al.
Read abstract…
After a thorough discussion of the epidemiologic, experimental, and other relevant data, the working group of International Agency for Research on Cancer (IARC) concluded in 2004 that formaldheyde is carcinogenic to humans. In the epidemiologic studies, there was suffcient evidence that formaldehyde causes nasopharyngeal cancer, “strong but not sufficient” evidence of leukemia, and limited evidence of sinonasal cancer. Molecular characterization of morphologic changes requires exquisite tissue morphology and DNA and RNA preservation; however, formaldehyde usually result in fragmented RNA. Aims: to optimize molecular analyses on fixed tissues, we assessed morphologic, immunogenicity, DNA and RNA integrity in human thyroid from non tumoral and tumoral pathology when specimens were fixed for different time course in several non-formalin fixatives.

Comparison of formalin and FineFIX in preserving DNA material in small biopsies

(Ref. RE-008)

Pathology International
Fatemeh Mahjoub et al.
Read abstract…
Formalin is a routine fixative in surgical pathology wards. Although morphological assessment is very desirable in formalin-fixed materials, DNA and RNA are not preserved well and molecular studies cannot be performed with reliable results. Biopsies and surgical specimens, as well as post mortem tissue samples, represent an important resource, especially for studies of molecular epidemiology, rare disease, neuropathology, and case studies with very long follow-up periods. A major advantage of using paraffin-embedded material is that it is easier to collect tissues from already existing archives in comparison to frozen fresh tissues that need a specific sample collection, with dedicated spaces and specific equipment. Because formalin fixation results in poor preservation of DNA and RNA, there are efforts to introduce new molecular friendly fixatives by some investigators. Recently a new fixative named FineFIX, which is ethanol based, has been introduced (Milestone, Sorisole, Italy) but few published articles exist about its advantages in improving molecular analysis. Nowadays many small samples are taken during endoscopy or needle biopsies and sent to pathology laboratories, and these may be the only available specimen for future molecular studies.We found no published data on DNA studies in small samples, so we decided to examine this fixative at a molecular level and compare it with formalin in small biopsies.

Morphological quality and nucleic acid preservation in cytopathology

(Ref. RE-009)

Journal of Clinical Pathology
Alessia Gazziero et al.
Read abstract…
Aim: Fixation is a chemical or physical procedure to prevent the degradation of protein and tissue morphology. To optimize molecular analyses on archival tissues, it is essential that fixation preserves morphology along with protein epitopes and DNA/RNA integrity. Methods: We employed a new formalin-free alcoholic-based fixative, named FineFix®, in 15 cases of serous effusions and 38 cases of Fine-Needle Aspirates, in order to evaluate the cellular morphology, and the quality of nucleic acids. Results: The cyto-morphology of the cellular component of effusions and Fine-Needle Aspirates obtained with FineFix® fixation was similar to that obtained with the matched formalin-fixed counterpart. Immunocytochemistry showed comparable results with the traditional fixative, but the formalin-free fixation preserved higher molecular weight DNA and RNA as demonstrated by successful PCR of large amplification products of more 2000 bp. Conclusions: The formalin-free fixative produced not only satisfactory results for immunocytochemistry on cytological smears and cell blocks, but also excellent preservation of DNA/RNA that can be efficiently employed also for sophisticated molecular techniques.

Two-Year Experience of a Formalin-Free Preservation Of The Human Organs and Total Corpses For Anatomical Dissection in Kaunas University of Medicine

(Ref. RE-010)

Institute of Anatomy, University of Medicine, Kaunas, Lithuania
V. Gedrimas et al.
Read abstract…
According to EU norms for workplace air (DIN EN 689: 1994-04-E), no inhalation of dangerous vapours from chemical agents utilizing routinely for embalming of organs and corpses in the human anatomy is allowed. Therefore, a formalin-free preservation of the human organs and total corpses for anatomical dissertation has been launched in Kaunas University of Medicine since 2007. The aim of the study is to prospect a suitability of the commercially available FineFix fixative for preservation of dissecting material in laboratories of study of the human anatomy.

An Initial Experience with Rapid Microwave Processing in the One-Stop Breast Clinic

(Ref. RE-011)

World Journal of Surgery
Edward Parkin et al.
Read abstract…
Background: Rapid microwave processing allows core biopsy results to be obtained within a 3 to 4 h time period. This study was designed to compare the accuracy and reporting time of microwave-processed breast biopsies with samples processed using traditional methods. Methods: Concordance of the preo perative biopsy report with post operative histology for tumor type, grade, and detection of lymphovascular invasion was recorded for both techniques. Also reviewed were the time taken between day of biopsy and day of reporting, waiting time between biopsy and surgery, and number of preoperative out patient appointments. Results: in the microwave-processed group (MG; n=43), there was a 92% concordance rate between preoperative biopsy and postoperative histology for tumor type. In the traditional group (TG; n=43), i twas 80% (P[0.05).For tumor grade, there was a concordance rate of 64% in MG and 93%in TG (P>0.05). For detection of lymphovascular invasion, there was agreement in 88% of cases in MG and 67% in TG (P>0.05). Twenty-five patients from MG were informed of their diagnosis on the day of biopsy. There was no difference in waiting time from biopsy to surgery or number of preoperative out patient appointments between MG and TG (P>0.05). Conclusions: Microwave processing allows safe and accurate immediate histological reporting. As a result, surgical management can be planned at the initial outpatient consultation.

Alternativas al formol como fijador de piezas y tejidos anatómicos

(Ref. RE-012)

Libro Blanco de la Anatomía Patológica en España
José Antonio Giménez Mas et al.
Read abstract…
El patólogo está tan familiarizado con el formol, como el fumador con el tabaco. Durante muchos años se ha subestimado el potencial lesivo de ambos carcinógenos lo que propició una posición de tolerancia y aceptación al tiempo que se mantenía una prolongada controversia sobre su efecto carcinógeno que finalmente la ciencia concluyó proporcionando suficientes evidencias. Basado en ellas, numerosos países han legislado para proteger a trabajadores y consumidores de la exposición a ambos agentes carcinógenos. Sin embargo, a pesar de su carácter irritante y estar clasificado como cancerígeno (de categoría 3 en la Unión Europea, de categoría 2 según el Reglamento (CE) 1272/2008 y de categoría 1 según la Agencia Internacional de Investigación del Cáncer o IARC (International Agency for Research on Cancer, World Health Organization), el formaldehído sigue siendo una de las sustancias más utilizadas en los centros sanitarios. Se ha recomendado eliminar su uso, si ello es posible, o si no reducir la exposición tomando las correspondientes medidas de corrección para lograr una disminución de sus niveles ambientales. En el LIBRO BLANCO de 2009, Córdoba y colaboradores ya alertaban del riesgo del formol y de las medidas de prevención necesarias. En este estudio pretendemos insistir en la necesaria concienciación de los profesionales expuestos y proponemos avanzar hacia la eliminación o limitación progresiva del formol en los servicios de anatomía patológica mediante el uso de los posibles sustitutos disponibles. Tomando el formol como gold estándar se ha comparado su capacidad de preservación tisular y penetración, así como la respuesta de los tejidos a determinadas tinciones especiales e inmunohistoquímicas, con tres fijadores propuestos como alternativas a la fijación formólica convencional. Igualmente, y como necesaria consecuencia se han analizado los riesgos laborales y medioambientales de los sustitutos así como la gestión de sus residuos.

Comparison of formalin-free tissue fixatives: a proteomic study testing their application for routine pathology and research

(Ref. RE-013)

Archives of Pathology & Laboratory Medicine
Hannelore Kothmaier et al.
Read abstract…
Context: Formalin-fixed, paraffin-embedded tissue is the routine processing method for diagnostics practiced in pathology departments worldwide. Objective: To determine the potential value of non-cross-linking, formalin-free tissue fixation for diagnostics in pathology and proteomic investigations. Design: We tested 3 commercially available, formalin-free tissue fixatives-FineFIX, RCL2, and HOPE-in lung cancer specimens from 10 patients. The fixatives were evaluated for their effects on tissue morphology, protein recovery, and immunoreactivity for a selected panel of proteins differently expressed in lung cancer, using immunohistochemistry and Western blotting. Results: Tumor-cell analysis with hematoxylin-eosin worked equally well for all tested fixatives when compared with the standard formalin-fixed, paraffin-embedded procedure. Movat pentachrome stains showed comparable results for the different matrices and cellular proteins analyzed. The RCL2 (P = .01) and HOPE fixatives (P = .03) improved protein recovery when compared with formalin-fixed, paraffin-embedded or frozen tissues. Our data clearly show that the fixatives evaluated influenced immunoreactivity to matched, formalin-fixed, paraffin-embedded lung cancer tissue. In particular, membrane-bound proteins, such as epidermal growth factor receptor EGFR, can be detected more efficiently by immunohistochemistry and Western blotting. Conclusion: We have demonstrated that formalin-free fixatives have the potential in routine pathology and research to replace formalin in histomorphology and protein preservation.

Formaldehyde Substitute Fixatives

(Ref. RE-014)

American Journal of Clinical Pathology
Cathy B. Moelans et al.
Read abstract…
Because formaldehyde is toxic and creates crosslinks that may hinder immunohistochemical studies, we tested 3 new cross-linking (F-Solv [Adamas, Rhenen, the Netherlands]) and non–cross-linking (FineFIX [Milestone, Bergamo, Italy] and RCL2 [Alphelys, Plaisir, France]) alcohol-based fixatives for routine staining in comparison with neutral buffered formalin (NBF) as the “gold standard.” Fresh tissue samples were divided into 4 equal pieces and fixed in all fixatives for varying times. After paraffin embedding, H&E staining, 7 common histochemical stains, and 9 common immunohistochemical stains were performed. RCL2 fixation resulted in soft and slippery tissue, causing sectioning difficulties. F-Solv and FineFIX led to partial tissue disintegration during fixation. F-Solv performed morphologically similar to NBF but needed considerable protocol adjustments before being applicable in daily histologic and immunohistochemical practice. FineFIX did not necessitate major protocol changes but caused shrinkage artifacts, degranulation, and lysis of RBCs. RCL2 generated morphologically overall good results without major protocol changes but caused pigment deposition, degranulation, and RBC lysis. The alcohol-based fixatives had positive and negative attributes and environmental drawbacks, and none was overall comparable to NBF with regard to macroscopy, morphologic evaluation, and immunohistochemical studies.

Evaluation of two commercial and three home made fixatives for the substitution of formalin:a formaldehyde–free laboratory is possible

(Ref. RE-015)

Environmental Health
Cristina Zanini et al.
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Background: Formaldehyde (HCHO) is a gas (available as a 37% concentrated solution, stabilized with methanol). The 10% dilution (approximately 4% formaldehyde) has been used as a fixative since the end of the 19th century. Alternative fixatives are also commercially available or may be prepared in-house in laboratories. Statements by the IARC, along with other USA agencies (CalEPA, RoC/NTP) on the carcinogenicity of formaldehyde for humans renders its substitution in Pathology Departments necessary since the annual use of formalin may exceed 3,500 liters for a medium-large laboratory. To achieve a“formalin-free laboratory” we tested straightforward-to-make fixatives along with registered reagents offered as formalin substitutes. Methods: More than two hundreds specimens were fixed in parallel within laboratory made fixatives PAGA (Polyethylenglycol, ethylAlcohol, Glycerol, Aceticacid), two zinc-based fixatives (ZBF,Z7), and commercially available alternatives (RCL2 and CellBlock). Tissue microarrays were used for morphological and immunohistochemical comparison. Extraction of RNA was carried out to evaluate preservation of nucleic acids. Results: Differences compared to formalin fixation were evident in alcohol-based fixatives, mainly restricted to higher stain affinity and considerable tissues hrinkage. Conversely, nuclear detail was superior with these alcohol-based formulas compared to formalin orglyoxale-based recipes. RNA extraction was superior for Z7, PAGA and RCL2 with regard to concentration but relatively comparable regarding quality. Conclusions: Abolition of the human carcinogen formaldehyde from pathology laboratories is possible even in contexts where by commercial alternatives to formalin are unavailable or are too expensive for routine use, and aspiration devices are lacking or not adequately serviced. The use of known formulations, possibly with simple and not-noxious (“alimentary grade”) constituents, comparable with registered proprietary products, may expand the search for the ideal fixative combining satisfactory morphology with improved preservation of nucleic acids and proteins as well as being easy and safe to dispose of. Keywords:Formalintoxicity, Alternative fixatives, Tissue microarray, RNAextraction

Preliminary testing of alternative fixatives to formalin for preservation of marine macrozoobenthos samples

(Ref. RE-016)

Biologia Marina Mediterranea
F. Oselladore et al.
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Three non-formalin-based fixatives (FineFIX, Glyoxal and Ethanol) to preserve marine macrozoobenthos samples were compared to find a suitable substitute to Formalin. Solutions were tested on Crustaceans and Polychaetes and evaluated according to specific time windows: 7, 15, 30, 60, 120 and 180 days. The considered variables included: color, wet weight, stiffness and resistance to shaking. Best results with Polychaetes were obtained using 40% FineFIX diluited in 60% Ethanol, while no significative differences were observed with Crustaceans using all the tested solutions.

The effect of the alternative solutions to formaldehyde and xylene on tissue processing

(Ref. RE-017)

Indian Journal of Pathology & Microbiology
Ilgin Aydin et al.
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Introduction and aim: To assess the impact of new alternative solutions to formaldehyde and xylene on tissue processing, 13 different tissue processings were designed and performed on thirteen different tissues by using five different fixatives (formaldehyde, Glyo-Fixx®, FineFix®, Cell-block®, Green-Fix®) and four different clearing agents (xylene, Sub-X®, Bio-clear®, Shandon Xylene Substitute®). Materials and methods: Hematoxylin and Eosine stained sections were compared by using qualitative histomorphological criterions. Histochemical and immunohistochemical (IHC) staining results were compared with qualitative and quantitative data obtained by a computer program, respectively. Tissue sections were tested for the availability of chromogenic in situ hybridization, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) extraction, and DNA quality by polymerase chain reaction. Results: The quality of sections was well for all tissue processings. All alternative solutions were suitable for histochemistry. IHC staining results showed that alternative solutions that contain glyoxal as active agent need optimization for this application. The clearance of signals with chromogenic in situ hybridization were nearly same and well for all tissue samples. Furthermore, tissue processes that do not contain formaldehyde were found to be superior on preservation of nucleic acids. Conclusion: Formaldehyde-free fixatives and alternative clearing agents have potential in routine pathology and research to replace formaldehyde and xylene.

Analysis of the effect of various decalcification agents on the quantity and quality of nucleic acid recovered from bone biopsies

(Ref. RE-018)

Annals of Diagnostic Pathology
Veena M Singh et al.
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Molecular studies are part of standard care for cancer patients. Bone, a common and sometimes sole site of metastasis, requires decalcification for morphological examination. Many commonly used decalcification agents contain strong acids that degrade nucleic acids. The paradigm shift in oncology, with biomarker targeted therapy and gene expression profiling analysis, requires sufficient nucleic acid recovery from bone biopsy specimens. We systematically studied the effects of a spectrum of decalcification agents on the quantity and quality of RNA and DNA recovered from bone biopsies. Multiple bone biopsies of similar size and cellularity were fixed in 10% neutral-buffered formalin, randomized to various decalcification agents for 2 hours then processed, and embedded. Tissue lysates were obtained from unstained sections and nucleic acid isolated. DNA and RNA were quantified. Assessment of DNA and RNA integrity was accomplished by comparison of the average cycle threshold by polymerase chain reaction of selected housekeeping genes for each agent. Results were then analyzed by 2-sample t test. There was a significant decrease in both DNA and RNA yield and integrity with strong acids (hydrochloric, nitric) vs 14% EDTA and formic acid. DNA yield was (mean nanograms) 6.15 vs 68.68 (P<.001) and RNA was (mean nanograms) 121.53 vs 288.89 (P=.003), respectively. DNA integrity (mean cycle threshold) was 35.79 vs 30.16 (P<.001), and RNA was 33.03 vs 26.5 (P<.001), respectively. Decalcification of bone biopsies with EDTA or formic acid agents was associated with a significant improvement in recovered nucleic acid quantity and quality.

Partial Oxygen Pressure Affects the Expression of Prognostic Biomarkers HIF-1 Alpha, Ki67, and CK20 in the Microenvironment of Colorectal Cancer Tissue

(Ref. RE-019)

Oxidative Medicine and Cellular Longevity
Lirong Zhang et al.
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Hypoxia is prognostically important in colorectal cancer (CRC) therapy. Partial oxygen pressure (pO2) is an important parameter of hypoxia. The correlation between pO2 levels and expression levels of prognostic biomarkers was measured in CRC tissues. Human CRC tissues were collected and pO2 levels were measured by OxyLite. Three methods for tissue fixation were compared, including formalin, Finefix, and Finefix-plus-microwave. Immunohistochemistry (IHC) staining was conducted by using the avidin-biotin complex technique for detecting the antibodies to hypoxia inducible factor-1 (HIF-1) alpha, cytokeratin 20 (CK20), and cell proliferation factor Ki67. The levels of pO2 were negatively associated with the size of CRC tissues. Finefix-plus-microwave fixation has the potential to replace formalin. Additionally, microwave treatment improved Finefix performance in tissue fixation and protein preservation. The percentage of positive cells and gray values of HIF-1 alpha, CK20, and Ki67 were associated with CRC development (P < 0.05). The levels of pO2 were positively related with the gray values of Ki67 and negatively related with the values of HIF-1 alpha and CK20 (P < 0.05). Thus, the levels of microenvironmental pO2 affect the expression of predictive biomarkers HIF-1 alpha, CK20, and Ki67 in the development of CRC tissues.

EDTA-based decalcification of bone and bone Marrow – ideal tool for protein and nucleic acid preservation – a pilot study

(Ref. RE-020)

Memorial Sloan-Kettering Cancer Center, New York, USA
Peter Ntiamoah et al.
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Bone and bone marrow biopsies are performed to diagnose diseases such as hematologic malignancies, primary bone tumors and metastatic tumors. In the era of increasing molecular testing for diagnosis and targeted therapeutics, preservation of nucleic acids and proteins are necessary and is required for the best patient care. For routine processing of bone specimens decalcification with inorganic acids (nitric acid or HCl) is used. Acid based methods allow fast turnaround times, but, the procedure restricts use of the tissue for molecular diagnosis by damaging DNA, RNA and is not suitable for some protein assays. To address this we undertook a pilot study using 10% EDTA decalcification solution, pH 7.2, as an alternate source for decalcification.

Mohs and the benefits of new embedding and staining systems

(Ref. RE-021)

Pathology in Practice
Guy Orchards
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Guy Orchards reports on preliminary work to assess the benefits of the latest equipment designed to facilitate frozen section preparation and staining in a laboratory supporting a Mohs micrographic surgery service.

Accelerated Tissue Processing With Minimal Formalin Fixation Time for 9-Gauge Vacuum-Assisted Breast Biopsy Specimens

(Ref. RE-022)

American Journal of Clinical Pathology
Joris P. Bulte et al.
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Objectives: Vacuum-assisted biopsy (VAB) of the breast seems unsuitable for rapid processing due to large size. We tested microwave-based acceleration. Methods: As a proof-of-principle study, 9-gauge VAB specimens were taken from eight mastectomy specimens. Forty-two biopsy specimens were processed. Quality of H&E was evaluated in 84 slides, and estrogen receptor (ER), progesterone receptor (PR), E-cadherin, and human epidermal growth factor receptor 2 (HER2) stains were evaluated in six slides. Preoperative biopsy specimens were used as a control. Results: Diagnostic quality of H&E slides was good in 87%, reasonable in 12%, and low in 1%. Quality of E-cadherin was good in 75% and reasonable in 25%. Quality of ER was good in 83% and reasonable in 17%. PR and both HER2 immunohistochemistry and fluorescence in situ hybridization were good in all slides. Quality of experimental slides was similar to control slides. Conclusions: Nine-gauge VAB specimens can be processed within 4 hours. Slides are suitable for all routine pathologic stains. This enables a same-day diagnosis.

Sample Handling

Under-vacuum preservation of tissues to be transferred to pathology labs

(Ref. SH-001)

Department of Biomedical Sciences and Oncology, University of Turin, Italy
G. Bussolati et al.
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Problem: • Formalin (4% Formaldehyde) is used worldwide as a fixative and preserver. • Formalin is toxic, allergenic and a carcinogen (IARC, 2006). • Surgical specimens to be processed for histopathological diagnosis are routinely immersed in Formalin, then transferred to Pathology labs. Aim: A safe formalin-free procedure for transferring tissues to Pathology Labs.

Evaluation of tissue preservation using a vacuum-based refrigeration system for specimen transfer from theatre to laboratory

(Ref. SH-002)

Department of Pathology, Royal Group of Hospitals Trust, Belfast, Northern Ireland
DP Boyle et al.
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Specimen preservation is essential to allow adequate morphological appraisal and successful application of immunohistochemistry. Traditionally immersion in formalin is ideal for morphology and immunohistochemistry but does not permit collection of fresh frozen tissue, the ideal for research purposes and biobanking. We evaluated a novel system for handling fresh specimens (TissueSAFE) which employs cacuum sealing and storage at 4°C for transfer to the pathology laboratory. Tissue slices from colectomy specimens were vacuum sealed using TissueSAFE and stored at 4°C prior to sampling mucosal, nodal and tumor tissue at subsequent time points (up to 72 hours) for snap freezing. In addition, composite paraffin blocks were generated at the same time points following an additional 24 hrs in formalin. H&E staining and immunostaining using a range of antobodies was applied to the paraffin blocks and DNA was extracted from the frozen tissue samples for comparison between times in TissueSAFE. There was no appreciable difference in morphology or immunohistochemical staining between any of the samples for any of the antibodies assessed. Extracted DNA quantities and quality were preserved with no depreciation with time in TissueSAFE. In conlcusion, TissueSAFE preserves tissue for morphological examination, immunohistochemistry and DNA extraction for up to at least 72 hours.

Vacuum-based preservation of surgical specimens: an environmentally-safe step towards a formalin-free hospital

(Ref. SH-003)

Science of the Total Environment
Cinzia Di Novi et al.
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Formalin as a fixative has no practical substitutes, but is toxic and potentially carcinogenic, so caution of its use in hospitals and elsewhere is mandatory. In our hospital, preservation of surgical specimens into formalin to be transferred to pathology labs was replaced by under-vacuum sealing (UVS) tissues into plastic bags and preservation at 4 degrees C until transfer. Data analysis showed UVS processing to be superior in terms of staff satisfaction and of gross anatomic preservation; no problems in terms of technical feasibility or histopathologic preservation were encountered. Formalin was confined to pathology labs while its use on hospital premises was vastly reduced.

Traslado de muestras quirúrgicas desde quirófano al Servicio de Anatomía Patológica. Intraoperatoria “versus” Sistema de alto nivel de vacío

(Ref. SH-004)

Congreso Red Nacional de Biobancos, Tarragona, Spain
R. Martínez Marín et al.
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Introducción: La llegada de muestras a un biobanco en determinados hospitales, suele ser un peregrinaje no exento de dificultades, si a todo eso le añadimos que hay hospitales que no están muy involucrados en la toma de muestra para biobancos, el conseguirlas es todavía una batalla considerable. Cuando el biobanco como concepto de servicios comunes inició su andadura la dinámica de guardar muestras para biobancos no estaba muy extendida, más allá de colecciones par culares. A su vez, u lizar conceptos como “trátese como intraoperatoria“ todavía creaba más ansiedad tanto para emisor como para el receptor de la muestra. Asimismo había que asimilar hacer un ”hueco” en la ac vidad diaria, teniendo en cuenta la importancia de la preservación de las muestras para estudios gené cos, moleculares, cul vos, etc.

Evaluation of tissue preservation of materials stored at 4°C with TissueSAFE system after the examination and macroscopic sampling

(Ref. SH-005)

Divisione di Anatomia Patologica e Medicina di Laboratorio Istituto Europeo di Oncologia Milano (IEO)
M. Manzotti et al.
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Maintenance of tissue features is essential for the evaluation of the biological parameters of neoplasms, for diagnosis and for further immunohistochemical investigations and studies of molecular biology. The use of formalin at 4% has traditionally dominated all phasesof the transportation process, of fixation and storage of surgical specimens. In the last few years a new system allows transportation of refrigerated (4°C) samples, that are put under-vacuum and not-fixed: the systems is called TissueSAFE (Milestone, Bergamo, Italy). The advantage of this system is the elimination of part of the formalin required for the transportation/fixation of surgical specimens and it allows thecontrol of the fixation time. Unlike the majority of other pathological anatomy services, at IEO over 95% of surgical specimens are immediately transferred to the grossing table, sampled fresh and subsequently preserved in formalin, until completion of the diagnostic process. In order to reduce the use of formalin, we wanted to evaluate the state of preservation of tissues and, in particular, DNA and RNA of specimens stored in refrigerator at 4°C with TissueSAFE system.

Keeping your TissueSAFE (Equipment Trial)

(Ref. SH-006)

Anatomical Pathology, Prince Charles Hospital, Chermside, Brisbane, Australia
David Butler
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Aims of the study: – determine if formalin use could be significantly reduced in operating theatres without adverse effects on surgical specimens – determine the suitability of existing courier arrangements for a formaline free preservation system

RNA preservation in FFPE tissues requires pre-analytical standardized processing

(Ref. SH-007)

Department Biomedical Sciences and Human Oncology, University of Turin, Italy
Gianni Bussolati
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Modalities of handling of surgical specimens need standardization in order to guarantee optimal preservation structure, protein antigenicity and nucleic acid sequences. For the transfer of Surgical specimens to Pathology laboratories, two types of modalities are currently practiced worldwide, according to local conditions and habits: A) Tissues are transferred fresh, B Tissues are immersed in Formalin containers. Preservation of RNA for Gene Expression profiling is notoriously poor in Formalin Fixed Paraffin Embedded ( FFPE) tissues, thus hampering extended gene sequencing in routinely processed histo-pathological tissues.

Guide de bonnes pratiques émis par le groupe Sénopath pour l’optimisation des prélèvements en pathologie mammaire

(Ref. SH-008)

Recommandations Sénopath
Senopath group, France
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Ces recommandations ont été élaborées par un groupe pluridisciplinaire composé de professionnels impliqués dans la prise en charge des cancers du sein dans le but d’homogénéiser et d’optimiser la gestion des prélèvements tissulaires (biopsies et pièces opératoires). Ce travail s’inscrit dans le cadre d’une démarche qualité, soutenue par le réseau ONCOMIP et le Comité Sein de l’IUC.

Vacuum-based preservation of colorectal cancer specimens: a comparison with formalin fixation

(Ref. SH-009)

Department of Pathology, Saint-Antoine Hospital, AP-HP, Medical School Pierre et Marie Curie, Paris, France
Jean-François Fléjou et al.
Read abstract…
In Pathology, an alternative to immediate fixation in neutral buffered formalin(NBF) is vacuum sealing and cooling (VSC) (“TissueSAFE”system).There have been few evaluation of VCS. Tissues that have been processed with VCS are not only used for standard morphological techniques,but also for molecular tests, mandatory in various types of solid tumours, including colorectal cancer.

Vacuum Packed Transport maintains the RNA quality

(Ref. SH-010)

ARC-NET applied research on cancer, Verona – Italy
Martina Farronato et al.
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Prolonged transport time and processing delays of tissue specimens affect RNA quality. The aim of this study was to test the impact of storing tissue samples under vacuum condition prior to processing on RNA integrity. We first tested the integrity of RNA extracted from 12 samples of murine tissue from the strain of nude mice Swiss-nu/nu. The results obtained from murine samples were then validated using samples of normal tissue from patients who underwent surgical resection for adenocarcinoma of the pancreas.

Vacuum sealing and cooling for a safe transfer of tissue from operating theatre to laboratory

(Ref. SH-011)

University of Turin, Italy
G. Bussolati et al.
Read abstract…
Background: the pre-fixation “ischemia” time represents a potentially dangerous step affecting preservation quality of both structure and tissue components (proteins, nucleic acids). While best practice requires immediate transfer of surgical specimens from theatres to Pathology laboratories, this is often impratical for structural reasons and a series of alternatives have been devised. Most common worlwide is the transfer of specimens in formalin-filled boxes, a practice which implies serveral drawbacks, both for tissue preservation and local environment.

Histologic Validation of Vacuum Sealed Formalin-Free Tissue Preservation and Transport System

(Ref. SH-012)

Pathology & Laboratory Medicine, HenryFord Health System, Detroit, Michigan
Michael Dib PA et al.
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Human specimens for pathologic examination are routinely fixed in formalin, a process that is over 100 years old. This potentially exposes healthcare personnel to potentially toxic and critical exposure to the class 1 carcinogen, formaldehyde. A formalin spill in the operating room (OR) or laboratory must be contained as a hazardous material and may present a major health hazard. It is our goal to redesign a work process so that formalin is removed from hospital OR’s for the purpose of fixation and transportation of human specimens to the Anatomic Pathology Core Laboratory of the Henry Ford Health System, Detroit, Michigan.

Analyse qualitative et quantitative d’échantillons tissulaires par le procédé TissueSAFE en vue d’analyses morphologiques et moléculaires. Evaluation sur une série de 10 cas.

(Ref. SH-013)

Institut Curie, Paris, France
Martial Caly et al.
Read abstract…
La préservation de l’intégrité tissulaire est primordiale pourgarantirnonseulementundiagnostic de qualitémais également les conditions optimales dédiéesàl’IHCetauxextractionsd’acidesnucléiques. Dans notre expérience, nous constatons parfois la difficulté de réaliser systématiquement les prélèvementsextemporanésdetissufraisnécessaires auxanalyses, telsque lacryopréservationalimentant notreCRB. En effet, certaines pièces anatomiques chirurgicalessont reçuesaulaboratoiredePathologie déjà fixées. Pour palier ce type de problèmes, l’automateTissueSafe®dechezMilestoneproposede conserver le tissu frais plusieurs heures, sous conditiondevideetà4°C.Cedélaidepriseencharge autoriseainsiaulaboratoireuntraitementmaitrisédes pièces tissulaires et donc la réalisation de tous le prélèvementsnécessairesauxanalysesultérieures. Ceprocédépermet également dese libérer des dangersetcontraintes liésauformol. Ilarriveeneffet quelespièceschirurgicalessoient immergéesdansle fixateurauseinmêmedublocopératoire, endehors de toute enceinte spécifiquement dédiée à son utilisation.

Contrôle de la fixation des mastectomies par la procédure Tissue SAFE

(Ref. SH-014)

Institut de Pathologie de Paris, France
B. Poulet et al.
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La procédure Tissue SAFE permet une conservation des pièces opératoires sous vide et à 4°pendant 72 h sans altération morphologique et moléculaire des tissus. En pathologie mammaire, les pièces opératoires doivent être fixées: – moins d’une heure après leur extraction – pour une durée totale de plus de 24 H et de moins de 48 H – avec un pré-tranchage de 5 à 10 mm pour les grosses pièces permettant une bonne pénétration du fixateur (vitesse de pénétration du formol à 4 % à 20°= 1mm/heure) En activitélibérale, du fait de l’éloignement géographique des blocs opératoires des cabinets de pathologie, il est difficile de maîtriser le conditionnement des mastectomies avant leur fixation. La procédure Tissue SAFE peut être utilisée pour différer la fixation des mastectomies qui sont fixées au cabinet de pathologie dans des conditions contrôlées pendant une durée contrôlée.

Efficient stem cell isolation from under vacuum preserved tissue samples

(Ref. SH-015)

Organogenesis
Aldo Moggio et al.
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Different approaches for the isolation of stem/progenitor cells have been reported, including stem cell selection in stringent culture conditions. We evaluated the possibility of isolating human progenitor cells from surgical specimens preserved by under vacuum sealing and cooling, a clinical practice approached by several hospitals as alternative to formalin. Renal tissue samples (n = 20) maintained under vacuum from 6 to 48 h at 4°C were used to isolate human renal CD133+ progenitor cells. We obtained CD133+ progenitors from unsorted cells derived from disaggregated tissues from each sample. Phenotypic characterization as well as in vitro and in vivo differentiation of the obtained CD133+ lines showed results comparable with sorted CD133+ cells obtained from fresh tissue. These results indicate that the process of sealing under vacuum and cooling appears as a suitable tissue treatment to isolate hypoxia resistant cells, such as human stem/progenitor cells, and that this procedure can be exploited to render the extraction of stem cells from human samples more practical and feasible.

Critical Steps in Tissue Processing in Histopathology

(Ref. SH-016)

Recent Patents on DNA & Gene Sequences
Maria Comanescu et al.
Read abstract…
Histopathological diagnosis using Formalin-Fixed Paraffin Embedded (FFPE) tissues is essential for the prognostic and therapeutic management of cancer patients. Pathologists are being confronted with increasing demands, from both clinicians and patients, to provide immunophenotypic and gene expression data from FFPE tissues to allow the planning of personalized therapeutic regimens. Recent improvements in the protocols for pre-analysis processing of pathological tissues aim to better preserve cellular details and to conserve antigens and nucleic acid sequences. These developments have been recently patented. The international protocol for the transporting of surgical specimens from the surgical theatre to the pathology department is to immerse the specimen in formalin. The alternative method of sealing the specimens into bags under a vacuum and then cooling is a well-accepted and environmentally safe procedure that overcomes the many drawbacks linked to transfer in formalin. Importantly, RNA is notoriously poorly preserved in FFPE tissue. Due to this, successful procedures for the extraction of genetic information from archival tissues have been the object of several studies and patents. Novel molecular approaches for RT-qPCR and gene array analysis on FFPE tissues are presented here. Moreover, a major advance is reported in this study, the observation that tissue fixation in cold conditions allows a much better preservation of nucleic acid sequences.

Recommendation for breast surgical specimens’ collection and diagnosis

(Ref. SH-017)

Raccomandazioni GIPaM
GIPaM, Italian group of Pathologists for studies on Breast Pathology 
Read abstract…
Documento condiviso nell’ambito del GIPaM

Vacuum sealing for a safe transfer of tissue from operating theatre to laboratory using TissueSAFE Milestone apparatus

(Ref. SH-018)

Hospital Universitario Central De Asturias – Spain ESBB Congress Granada
Isidro-Marron, P et al.
Read abstract…
The pre-fixation “ischemia” time represents a potentially dangerous step affecting preservation tissue components (nucleic acids). We tested Under-Vacuum sealing (UVS) system TissuSAFE (Milestone) versus fresh tissue or preserved in RNA later.

A Collection of Primary Tissue Cultures of Tumors from Vacuum Packed and Cooled Surgical Specimens: A Feasibility Study

(Ref. SH-019)

Plos one
Laura Annaratone et al.
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Primary cultures represent an invaluable tool to set up functional experimental conditions; however, creation of tissue cultures from solid tumors is troublesome and often unproductive. Several features can affect the success rate of primary cultures, including technical issues from pre-analytical procedures employed in surgical theaters and pathology laboratories. We have recently introduced a new method of collection, transfer, and preservation of surgical specimens that requires immediate vacuum sealing of excised specimens at surgical theaters, followed by time-controlled transferring at 4°C to the pathology laboratory. Here we investigate the feasibility and performance of short-term primary cell cultures derived from vacuum packed and cooled (VPAC) preserved tissues. Tissue fragments were sampled from 52 surgical specimens of tumors larger than 2 cm for which surgical and VPAC times (the latter corresponding to cold ischemia time) were recorded. Cell viability was determined by trypan blue dye-exclusion assay and hematoxylin and eosin and immunohistochemical stainings were performed to appreciate morphological and immunophenotypical features of cultured cells. Cell viability showed a range of 84–100% in 44 out of 52 (85%) VPAC preserved tissues. Length of both surgical and VPAC times affected cell viability: the critical surgical time was set around 1 hour and 30 minutes, while cells preserved a good viability when kept for about 24 hours of vacuum at 4°C. Cells were maintained in culture for at least three passages. Immunocytochemistry confirmed the phenotype of distinct populations, that is, expression of cytokeratins in epithelioid cells and of vimentin in spindle cells. Our results suggest that VPAC preserved tissues may represent a reliable source for creation of primary cell cultures and that a careful monitoring of surgical and cold ischemia times fosters a good performance of primary tissue cultures.

Validation of Tissue Preservation Using a Vacuum Sealed Formalin-Free Transportation System

(Ref. SH-020)

Department of Pathology, Henry Ford Health System, Detroit, Michigan
Richard J. Zarbo et al.
Read abstract…
Background: Formalin fixing and transporting tissue specimens is a process over 100 years old that potentially exposes healthcare workers to toxic, carcinogenic formaldehyde. Formalin spilled in operating room (OR) or laboratory is a health hazard to be contained as a hazardous material. We seek to redesign this work process so formalin is removed from 4 hospitals in the process of transportation of human specimens to an Anatomic Pathology Core Laboratory. Design: TissueSAFE high vacuum biospecimen transfer system (Milestone Medical, Kalamazoo, MI) was evaluated for vacuum sealed, formalin-free transfer and storage in 3 validations: 1) Preservation of surgical specimens vacuum sealed and stored at 4°C and 7°C for 24, 48 and 72 hours compared to paired formalin fixed tissue; 2) Transport and storage of specimens sealed in OR of a community hospital 25 miles away at 4°C for 24 and 48 hours; 3) Preservation of surgical specimens sealed in FS Room adjacent to 30 ORs then transported at 4°C to the Core Lab within the same hospital, range of storage times 1-10 hours before tissue examination, fixation, processing and histologic assessment. All slides were H+E stained and evaluated by pathologists using a 3 part scale of Acceptable for Diagnosis, Inferior Quality for Diagnosis or Unacceptable for Diagnosis. Results: SCHEME #1: 9 cases, 7 tissue types (50 blocks): liver, uterus, ovary, lymph node, leg, lung, fatty pannus. All were Acceptable for Diagnosis except for 4 blocks of liver that were under processed and coded as Inferior Quality for Diagnosis . SCHEME #2: 9 cases, 8 tissue types (56 blocks): stomach, gallbladder, placenta, fallopian tube, uterus, thyroid, bowel, fistula. All were Acceptable for Diagnosis by histologic evaluation at 1 and 2 days storage under vacuum in the cold. SCHEME #3: 30 cases, 16 tissue types, (164 blocks): lung, kidney, stomach, uterus, colon, small intestine, gallbladder, thyroid, brain, tonsil, skin, TURBT, TURP, hemorrhoid, appendix, arterial plaque. All but 2 cases were Acceptable for Diagnosis with 1 TURBT (5 hour storage) and kidney (2.5 hour storage) deemed Inferior Quality for Diagnosis due to “air-drying” and lack of crisp cytologic tumor detail, respectively. Conclusions: Vacuum sealed tissue held at 4°C are preserved for histologic assessment when held in that state up to 48 hours. The nature of the 2 histologically inferior exceptions with low storage times require further study. TissueSAFE vacuum transport shows promise to provide a platform for designing formalin-free ORs and transport of tissue specimens to processing laboratories.

Validation of Vacuum-Based Refrigerated System for Biobanking Tissue Preservation: Analysis of Cellular Morphology, Protein Stability, and RNA Quality

(Ref. SH-021)

Biopreservation and Biobanking
Valentina Condelli et al.
Read abstract…
Biobanks of fresh, unfixed human normal and malignant tissues represent a valuable source for gene expression analysis in translational cancer research and molecular pathology. However, the success of molecular and cellular analysis in both clinical and translational research is strongly dependent on the collection, handling, storage, and quality control of fresh human tissue samples. The aim of this study was to evaluate an innovative vacuum-based refrigerated system, as a logistically feasible technology to increase the collection of tissue specimens, preserving the integrity of cellular and molecular components. We tested randomly-selected tissues stored under vacuum at 4°C by using endpoints important for research and diagnosis, including tissue morphology, epitope stability, and RNA integrity. Gene expression was evaluated by qualitative and quantitative RT analysis of selected housekeeping and tissue-specific genes. Tissue morphology and overall protein stability were generally well preserved, being compromised only in gallbladder tissue. By contrast, phosphoprotein and RNA analysis demonstrated a time-dependent degree of degradation, with progressive loss of stability from 24 to 72 hours. However, this reduction in RNA quality did not represent a limitation for successful expression analysis of selected genes. Indeed, a comparative qualitative and quantitative RT-PCR analysis showed that RNA extracted from tissues stored under vacuum is suitable for gene expression profiling, but requires highly sensitive technologies, such as quantitative RT-PCR. These data suggest that the refrigerated vacuum-based system represents a suitable and feasible technology for routine transport of fresh specimens from surgery to biobanks, thus increasing the opportunity to collect biospecimens.

Advances in Pathology Tissue Management Reduce Formalin Use, Improve Quality and Cut Costs

(Ref. SH-022)

DARK daily Laboratory and Pathology News
Mark Terry
Read abstract…
Formalin has been used as a tissue preservative in operating rooms and pathology laboratories for over one hundred years. Formalin is a solution of about 4% formaldehyde and water (known as 10% NBF) and is ubiquitous in clinical laboratories, pathology laboratories, and in operating rooms. It is an inexpensive reagent and effective at what it does. Unfortunately, it is hazardous. According to the U.S. Department of Labor’s Occupational Safety & Health Administration (“OSHA”), formalin/formaldehyde is a moderate fire and explosion hazard when exposed to heat or flame and when mixed with certain chemicals. More problematic are its health hazards. Formalin is highly irritating to the upper respiratory tract, eyes, and skin, and is a known carcinogen. “In humans, formaldehyde exposure has been associated with cancers of the lung, nasopharynx and oropharynx, and nasal passages.”1 It is also considered to be mutagenic. In addition to those problems, modern healthcare institutions often have multiple clinics, hospitals, operating rooms, and laboratories at separate geographic locations. This requires that surgical tissue samples, large or small, be transported over significant distances in formalin-filled containers, greatly increasing the risk of potentially dangerous formalin spills. This white paper will look at the risks of formalin use, trends in formalin use and disposal, and how laboratory and operating room workflow is affected by it. Alternate methods for storing and preserving pathology specimens will be introduced along with descriptions of changes to workflow, benefits in terms of healthcare worker safety, and the safety and economic benefits of these changes. Two case studies, one of a major metropolitan hospital that has adopted two systems that modify or eliminate traditional formalin usage, and the other a major hospital in Italy, will be presented.

Guidance for laboratories performing molecular pathology for cancer patients

(Ref. SH-023)

Journal of Clinical Pathology
Ian A. Cree et al.
Read abstract…
Molecular testing is becoming an important part of the diagnosis of any patient with cancer. The challenge to laboratories is to meet this need, using reliable methods and processes to ensure that patients receive a timely and accurate report on which their treatment will be based. The aim of this paper is to provide minimum requirements for the management of molecular pathology laboratories. This general guidance should be augmented by the specific guidance available for different tumour types and tests. Preanalytical considerations are important, and careful consideration of the way in which specimens are obtained and reach the laboratory is necessary. Sample receipt and handling follow standard operating procedures, but some alterations may be necessary if molecular testing is to be performed, for instance to control tissue fixation. DNA and RNA extraction can be standardised and should be checked for quality and quantity of output on a regular basis. The choice of analytical method(s) depends on clinical requirements, desired turnaround time, and expertise available. Internal quality control, regular internal audit of the whole testing process, laboratory accreditation, and continual participation in external quality assessment schemes are prerequisites for delivery of a reliable service. A molecular pathology report should accurately convey the information the clinician needs to treat the patient with sufficient information to allow for correct interpretation of the result. Molecular pathology is developing rapidly, and further detailed evidence-based recommendations are required for many of the topics covered here.

Del quirófano al biobanco: circuito de recogida de muestras de cancer de mama

(Ref. SH-024)

BioBanco en Red de la region Murcia (BIOBANC-MUR)
Alberto Martinez Carrasco et al.
Read abstract…
El establecimiento de circuitos de recogida de muestras estables e integrados en los propios del hospitales es esencial para asegurar la calidad de las muestras almacenadas en el biobanco. Para desarrollar este proceso es fundamental la colaboracion entre los servicios hospitalarios involucrados y el biobanco.

Guidelines for Anatomical Pathology Laboratories on traceability, collection, transport, storage and archiving of cells and tissues for diagnostic purposes (italian)

(Ref. SH-025)

Italian Ministry of Health
Read abstract…
Anatomic Pathology Guidelines

Implantaciòn de un circuito de preservation de muestras de trasplante hepático envasadas al vacío, en el Nodo Biobanco H. Virgen del Rocìo-IBiS

(Ref. SH-026)

Nodo Biobanco H. Virgen del Rocìo-IBiS, Sevilla, Spain
Robles-Frias MJ et al.
Read abstract…
La disponibilidad de muestras en fresco es fundamental para poder criopreservar tejidos a -80°C. Sin embargo, no es facil disponer de estas muestras en determinados circuitos quirurgicos (cirugia de trasplante, de fin de semana, nocturna). Por ello, es necesario recurrir a nuevos sistemas de preservacion que garanticen la conservacion de las muestras mantiendo sus caracteristicas morfologicas y moleculares, de cara a su uso en investigacion biomedica.

Molecular Validation Study of Nucleic Acids Extraction from Vacuum Sealed Surgical Pathology Specimens

(Ref. SH-027)

Henry Ford Hospital, Detroit, Michigan
Dhananjay A. Chitale et al.
Read abstract…
Background: Formalin is the universal fixative in surgical pathology laboratories world over, however, it is a highly toxic irritant and potentially carcinogenic. It requires diligent care in its usage. To eliminate formalin use for specimen transport to the laboratory, we placed the surgical specimens in plastic bags under-vacuum sealing. Our aim was to assess if this method of transport could be used routinely without compromisingintegrity of the nucleic acids in this molecular era. Design: 15 surgically resected specimens from 3 anatomic sites (5 colon, 5 lung, 5 uterus) were selected for this pilot study. Specimens were placed in plastic bags under-vacuum sealing and transported at 4°C. 4 normal tissue samples using a 5 mm skin biopsy punch were collected at 0, 1, 2, 3 hour in duplicate; one snap frozen at -80°C & the other processed as formalin fixed paraffin embedded tissues (FFPE) [60 fresh, 60FFPE]. Manual DNA column extraction protocol for genomic DNA isolation & total RNA isolation protocol to isolate total RNA were run. DNA/RNA quantities were evaluated by spectrophotometry. DNA integrity was assessed by amplification of commercial Control Size Ladder mix generating a series of ampliconsof 100, 200, 300, 400 base pairs (bp). RNA integrity was assessed by real-time quantitative reverse transcription PCR by measurement of expression of ?2 microglobulin (B2M) transcripts. Cycle threshold (Ct) values of 30 fresh & 30 for FFPE tissue samples were used with a cut-off of > 37.0 Ctfor acceptable RNA quality. Results: H & E stained sections from all the FFPE samples showed optimal histologic preservation. The yield of extracted DNA & RNA was adequate withgood purity for both fresh & FFPE tissues [A260/A280 OD ratios >1.10 (range DNA:1.10-2.11, RNA:1.32-2.73)]. Fresh tissues: Total recovery between 6.46ug to 238.51 ug, the amplified DNA yielded product sizes of 300 bp for 54/60 samples, 400 bp for 53/60 samples. RNA from all the samples was of good quality, with acceptable B2M amplification in 28/30 samples. FFPE tissues: Total recovery between 3.12 ugto 81.09ug, the amplified DNA yielded product sizes of 300 bp for 60/60 samples, 400 bp for 60/60 samples. RNA from all the samples was of good quality, with acceptable B2M amplification in 29/30 samples. Conclusions: Our data suggests that the DNA and RNA integrity is well preserved after 3 hours of vacuum sealing of the surgical specimens without formalin fixation. Vacuum-based preservation of surgical specimens is a viable, molecular friendly and environmentally-safe option for tissue transport to laboratory.

Papel de biobanc-MUR, biobanco en red de la region de Murcia, en la creacion de la cohorte NELA

(Ref. SH-028)

BioBanco de la region Murcia, Spain
Agustin Parra Montoya et al.
Read abstract…
La cohorte al nacimiento NELA se esablece, fundamentalmente, para estudiar el papel de factores geneticos, epigeneticos y ambientales durante el embarazo y los primeros anos de vida en el origen del asma, la enfermedad cronica mas frecuente en la infacia. BIOBANC-MUR es responsable del procesado y almacenamiento de las muestras biologicas recogidas en esta cohorte, asegurando su calidad y trazabilidad.

Pre-Analytics of Pathological Specimens in Oncology

(Ref. SH-029)

Springer
Manfred Dietel et al.
Read abstract…
Recent Results in Cancer Research

Preservacion del ARN de Placenta conservada mediante sistema de alto vacio TissueSAFE: estudio piloto

(Ref. SH-030)

BioBanco de la region Murcia, Spain
Sergio Abenza Camacho et al.
Read abstract…
Controlar las condiciones de recogida y almacenamiento de muestras biologicas obtenidas en estudios epidemiologicos es imprescindible para asegurar su calidad y la fiabilidad de posteriores resultados. El sistema TissueSAFE permire envasar tejidos al vacio en el momento de la obtencion y conservarlos a 4° C hasta su procesamiento. En el caso de la placenta, especialmente sensible a la degradacion, este sistema aseguraria la preservacion del ARN y otros metabolitos.

Reducing formalin exposure risks in the management of surgical specimens

(Ref. SH-031)

Pathology in practice
Karen Lester
Read abstract…
To combat concerns about the dangers posed by the use and transport of formalin solutions, histopathology staff in Sheffield explored an innovative biospecimen transfer system, as Karen Lester explains.

RNA Quality in Fresh-Frozen Gastrointestinal Tumor Specimens. Experiences from the Tumor and Healthy Tissue Bank TU Dresden

(Ref. SH-032)

Recent Results in Cancer Research – Springer
Silke Zeugner et al.
Read abstract…
The term “pre-analytics” summarizes all procedures concerned with specimen collection or processing as well as logistical aspects like transport or storage of tissue specimens. All or these variables as well as tissue-specific characteristics affect sample quality. While certain parameters like warm ischemia or tissue-specific characteristics cannot be changed, other parameters can be assessed and optimized. The aim of this study was to determine RNA quality by assessing the RIN values of specimens from different organs and to assess the influence of vacuum preservation. Samples from the GI tract, in general, appear to have lower RNA quality when compared to samples from other organ sites. This may be due to the digestive enzymes or bacterial colonization. Processing time in pathology does not significantly influence RNA quality. Tissue preservation with a vacuum sealer leads to preserved RNA quality over an extended period of time and offers a feasible alternative to minimize the influence of transport time into pathology.

Incorporación de tejidos en biobanco a través de TissueSAFE

(Ref. SH-033)

BioBanco Universitario de Malaga, Spain
Martin I. et al.
Read abstract…
En los Biobancos. La seguridad e integridad de las muestras es de suma importancia. Miles de muestras irremplazables por su valor tanto diagnostico como para uso en investigacion son recogidas y conservadas a diario en los hospitales. Ademas la prevencion de riesgos laborales es tambien un tema crucial.

Últimos avances en la evaluación de la calidad de los tejidos archivados en los biobancos

(Ref. SH-034)

Biobanco Hospitalario Virgen del Rocìo-IBiS
Villena C. et al.
Read abstract…
La calidad de las muestras tisulares puede estar afectada por factores pre-analiticos que suceden antes de ser extraidas del paciente (variables pre-adquisicion, como puede ser el tratamiento recibido por el paciente, el tiempo de isquemia caliente, etc.), y tras la extraccion hasta su analisis (post-adquisicion, como son el tiempo de isquemia fria, preservantes utilizados, tiempo de almacenamiento, modo de descongelacion, etc.). Todos estos factores pueden provocar cambios moleculares y celulares no deseados en las muestras que interferiran en la fiabilidad de los resultados analiticos posteriores objeto de estudio. Dado que la principal mision de los biobancos es proveer muestras de calidad a la comunidad cientifica, la Plataforma Red Nacional de Biobancos ( PRNB) constituyo una linea (L3) para el esyudio de la calidad y desarrollo de nuevos procedimientos para el procesamiento y almacenamiento de tejidos en el seno del programa I+D+i (P3), costituyendo el grupo cooperativo L3P3. Asì, el objetivo inmediato durante 2015 ha sido averiguar el estado del arte sobre los controles de calidad testados en muestras de tejido.

Applicability of Under Vacuum Fresh Tissue Sealing and Cooling to Omics Analysis of Tumor Tissues

(Ref. SH-035)

Biopreservation and biobanking
Silvia Veneroni et al.
Read abstract…
Context: Biobanks of frozen human normal and malignant tissues represent a valuable source for “omics” analysis in translational cancer research and molecular pathology. However, the success of molecular and cellular analysis strongly relies on the collection, handling, storage procedures, and quality control of fresh human tissue samples. Objective: We tested whether under vacuum storage (UVS) effectively preserves tissues during the time between surgery and storage for “omics” analyses. Design: Normal and matched tumor specimens, obtained from 16 breast, colon, or lung cancer patients and 5 independent mesenchymal tumors, were dissected within 20 minutes from surgical excision and divided in three to five aliquots; for each tissue sample, one aliquot was snap-frozen in liquid nitrogen (defined as baseline or T0 samples), and the other portions were sealed into plastic bags and kept at 4°C for 1, 24, 48, or 72 hours under vacuum and then frozen. The tissue and molecular preservation under vacuum was evaluated over time in terms of histomorphology, transcription (Illumina microarrays), protein (surface-enhanced laser desorption/ionization-time of flight/mass spectrometry and Western blot), and metabolic profile (nuclear magnetic resonance spectroscopy). Results: Tissue morphology, Mib-1, and vimentin immunostaining were preserved over time without signs of tissue degradation. Principal variance component analysis showed that time of storage had a minimal effect on gene expression or the proteome, but affected the preservation of some metabolites to a greater extent. UVS did not impact the RNA and protein integrity or specific phosphorylation sites on mTOR and STAT3. Measurement of metabolites revealed pronounced changes after 1 hour of storage. Conclusions: Our results show that UVS can preserve tissue specimens for histological, transcriptomic, and proteomic examinations up to 48 hours and possibly longer, whereas it has limitations for metabolomic applications.

Automatizacion, estandardizacion y seguridad en el manekìjo de muestras biologicas

(Ref. SH-036)

Complejo Hospitalario Universitario de Ourense
Maria Luisa Lopez Vega et al.
Read abstract…
A dia de hoy el fijador “Gold Standard” dentro de los laboratorios de Anatomia Patologica sigue siendo el formol. La Agencia Inrenacional para la Investigacion del Cancer lo ha clasificado como cancerigeno de categoria 1B, con la indicacion de peligro H350 y mutageno de categoria 2, con la indicacion de peligro H341 segun el reglamento UE 605/2014 CLP. Esta nueva clasificacion y la enrtrada en vigor de la normativa actual en enero de 2016, lleva a las autoridades sanitarias a investigar el modo de eliminar o minimizar su uso en el entorno de trabajo.

Diagnostic procedures for non-small-cell lung cancer (NSCLC): recommendations of the European Expert Group

(Ref. SH-037)

The British Medical Journal
Manfred Dietel et al.
Read abstract…
Background: There is currently no Europe-wide consensus on the appropriate preanalytical measures and workflow to optimise procedures for tissue-based molecular testing of non-small-cell lung cancer (NSCLC). To address this, a group of lung cancer experts (see list of authors) convened to discuss and propose standard operating procedures (SOPs) for NSCLC. Methods: Based on earlier meetings and scientific expertise on lung cancer, a multidisciplinary group meeting was aligned. The aim was to include all relevant aspects concerning NSCLC diagnosis. After careful consideration, the following topics were selected and each was reviewed by the experts: surgical resection and sampling; biopsy procedures for analysis; preanalytical and other variables affecting quality of tissue; tissue conservation; testing procedures for epidermal growth factor receptor, anaplastic lymphoma kinase and ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) in lung tissue and cytological specimens; as well as standardised reporting and quality control (QC). Finally, an optimal workflow was described. Results: Suggested optimal procedures and workflows are discussed in detail. The broad consensus was that the complex workflow presented can only be executed effectively by an interdisciplinary approach using a well-trained team. Conclusions: To optimise diagnosis and treatment of patients with NSCLC, it is essential to establish SOPs that are adaptable to the local situation. In addition, a continuous QC system and a local multidisciplinary tumour-type-oriented board are essential.

Towards a formalin-free hospital. Levels of 15-F2t-isoprostane and malondialdehyde in nurses from operating theaters

(Ref. SH-038)

Toxicology Research
Valeria Bellisario et al.
Read abstract…
Purpose: nurses are exposed to formaldehyde when managing surgical samples that are to be later transferred to histopathology. We evaluated the conditions favouring the risk of exposure to this toxic reagent and the effect of measures to prevent it. Methods: we conducted a cross-sectional study where 94 female workers were enrolled as being potentially exposed to formaldehyde. From each nurse were collected: (1) personal air-formaldehyde by a personal dosimeter (8 hours), (2) a standardized questionnaire, (3) a urine sample to test 15-F2t-isoprostane, malondialdehyde, cotinine. Results: the results indicate a marked difference related to the adoption of the under vacuum sealing procedure, as an alternative to formaldehyde for preserving tissues. Nurses using the under vacuum sealing system in the operating rooms are exposed to levels of formaldehyde 75% lower than those who do not use that system. Oxidative stress biomarkers (15-F2t-isoprostane, malondialdehyde) are significantly higher in nurses using formaldehyde (p < 0.001) and in the absence of the under vacuum sealing system (p = 0.027), in particular in those workers who use liquid formaldehyde in the operating theatre (p = 0.012). Conclusions: analysis of the biological biomarkers confirms a direct responsibility of air formaldehyde on the onset of oxidative stress while the use of the under vacuum sealing technique is associated with a significant reduction of the exposure to air-formaldehyde and redox status. Our findings can be useful to characterize the environmental health risk in operating theatres and to plan preventive measures such as the under vacuum sealing procedure.

Validation of Placental Preservation for Pathologic Examination Using the TissueSAFE Formalin-Free Vacuum-Sealing System

(Ref. SH-039)

Department of Pathology, Henry Ford Health System, Detroit, Michigan
Richard J. Zarbo et al.
Read abstract…
Background: Placentas are the unloved specimens of Labor and Delivery(L&D) and Pathology. Most are normal and not seen in Pathology but require safe systems for storage and disposal. Placentas that require pathologic evaluation present numerous challenges of non-standardized ex vivo time, tissue preservation conditions, timed exposure to formalinfixation, consumption of high volumes of formalin for proper fixation and expensive disposal of the specimen and associated bucket of bloody formalin as hazardous waste. To address these issues, we have validated a vacuum-sealed, formalin-free tissue handling system designed to originate from L&D with intent to control placenta preservation and transport to Pathology for histologic examination. Design: 8 placentas were transported fresh from L&D to Henry Ford Hospital Pathology. Each was dissected according to protocol with 3 standard sections for immediate formalin fixation. Placentas were then sealed under low vacuum with the TissueSAFE system (Milestone Medical, Kalamazoo, MI) and retrieved for dissection and formalin fixation after storage in vacuum-sealed bags at 4°C for 24 hours (8 cases), 48 hours (4 cases) and 72 hours (4 cases). H&E stained slides were independently assessed by 2 placental pathologists using a 3 part scheme of: 1) Adequate, 2) Less than optimal, or 3) Inadequate for histologic evaluation. This study was IRB-exempt. Results: All 102 H&E slides were assessed adequate for histologic evaluation. No degradation of histologic detail was noted between fresh, formalin fixed to delayed fixation after 72 hours of vacuum-sealed cold storage. Conclusions: This histologic validation of vacuum-sealed human placentas allows consideration of differently designed processes for tissue handling by caregivers and pathologists. Potential advantages are controlled preanalytic placenta preservation up to 3 days at refrigerator temperature. Controlled fixation of fresh sections from each preserved placenta markedly reduces the amount of formalin (approximately 1 gallon/placenta) ordinarily used to fix the entire placenta before gross examination. Reduction of formalin at the front-end process of initial fixation translates to reduced back-end disposal of formalin as expensive hazardous waste. Both reductions translate to financial savings to the healthcare system. In L&D practices, this may eliminate formalin from the unit. Additionally, most placentas require disposal and vacuum-sealing of individual specimens provides for a safe isolation and disposal avoiding a fetid waste receptacle in L&D.

L’importanza dell’immunoistochimica nella diagnosi del carcinoma polmonare e sua ricaduta nella terapia mirata

(Ref. SH-040)

Azienda Ospedaliera dei colli Monaldi-Cotugno-CTO, Napoli, Italy
Giuseppe Salatiello et al.
Read abstract…
Il carcinoma del polmone rappresenta la causa di morte più frequente di cancro al mondo e se ne diagnosticano circa 1,35 milioni all’anno. La classificazione attuale impiegata, proposta dall’OMS prevede come istotipi più frequenti gli Adenocarcinomi, ADC (40%) ed i carcinomi di tipo Squamoso, SQC (23%).

Reliable histopathological diagnoses

(Ref. SH-041)

International Innovation
Anna Sapino
Read abstract…
Professor Anna Sapino aims to improve the pre-analytical phases of tissue specimen management, thereby ensuring that correct diagnoses are made. Working in cancer pathology, she describes a novel method to prevent biomarker degradation

Nuevo sistema automático de llenado de formaldehído en el servicio de anatomía patológica

(Ref. SH-042)

Dpto Anatomía Patológica, Hospital Universitario Vall d’Hebrón, Barcelona, Spain
Gisela Modena Riba et al.
Read abstract…
A partir del día 1 de enero de 2016 los Laboratorios de Anatomía Patológica deben cumplir el RD 665/1997 de Prevención de Riesgos contra Agentes Cancerígenos para la Monitorización, Ventilación, Extracción y Control del Formaldehído. El Sistema automático de llenado de formaldehído SEALSAFE, evita vapores, derrames y minimiza el contacto directo del personal con el formaldehído. Las muestras pueden conservarse hasta un máximo de 72h sin formol garantizando una óptima conservación de la morfología, la antigenicidad, el ADN, ARN y las proteínas.

Fresh tissue handling pathway for the 100,000 Genomes Project

(Ref. SH-043)

Pathology in practice
NHS West Midlands Genomic Medicine Centre, England
Read abstract…
In order to optimise and standardise the handling of fresh tissue for The 100,000 Genomes Project, four partner trusts of the West Midlands Genomic Medicine Centre have adopted an innovative vacuum-packing solution.

A new archiving system for the brain biobank in Vigo (Spain)

(Ref. SH-044)

Biobanco del instituto de investigacion sanitaria galicia sur, Vigo, Spain
Olga Souto et al.
Read abstract…
The Biobank in Vigo has a collection of more than 200 cases of neurodegenerative brain diseases. For each case they have archived one frozen brain hemisphere and the other hemisphere fixed in formalin and used for neuropathology diagnosis. Until now, the excess of fixed tissues was archived following a traditional method that is storing them in a plastic container (Tupper). This container, despite it is practical, it’s not totally hermetic therefore it’s very much possible to have formalin leak (being formaldehyde irritant and human carcinogenic agent). – Storing tissues under vacuum is a largely used method to transport and manage cases and to control the formaldehyde exposition in the Anatomical Pathology service. – The Biobank in Vigo modified its cases handling system, substituting the plastic containers with vacuum bags system

Adaptation of hospital clinico universitario “Lozano Blesa” pathology service and other services involved, because new european regulation on classification, labeling and packaging of substances and mixtures with formaldehyde

(Ref. SH-045)

European Society of Pathology
Pérez L. et al.
Read abstract…
Recently, EU regulation on classification, labeling and packaging of substances and mixtures with formaldehyde, changed classification of this product, going from category 2 (H351, suspect to causes cancer), to category 1B (H350, carcinogenic). This force pathology services to adapt.

Biopathologie des carcinomes ovariens des stades précoces et avancés

(Ref. SH-046)

Gynécologie Obstétrique Fertilité & Sénologie
M. Devouassoux-Shisheboran et al.
Read abstract…
Objectives. Ovarian carcinomas represent a heterogeneous group of lesions with specific therapeutic management for each histological subtype. Thus, the correct histological diagnosis is mandatory. Material and methods. References were searched by PubMed from January 2000 to January 2018 and original articles in French and English literature were selected. Results and conclusions. In case of ovarian mass suspicious for cancer, a frozen section analysis may be proposed, if it could impact the surgical management. A positive histological diagnosis of ovarian carcinoma (type and grade) has to be rendered on histological (and not cytological) material before any chemotherapy with multiples and large sized biopsies. In case of needle biopsy, at least three fragments with needles > 16G are needed. Histological biopsies need to be formalin-fixed (4% formaldehyde) less than 1 h after resection and at least 6 hours fixation is mandatory for small size biopsies. Tissue transfer to pathological labs up to 48 hours under vacuum and at +4 8C (in case of large surgical specimens) may be an alternative. Gross examination should include the description of all specimens and their integrity, the site of the tumor and the dimension of all specimens and nodules. Multiples sampling is needed, including the capsule, the solid areas, at least 1 to 2 blocks per cm of tumor for mucinous lesions, the Fallopian tube in toto, at least 3 blocks on grossly normal omentum and one block on the largest omental nodule. WHO classification should be used to classify the carcinoma (type and grade), with the use of a panel of immunohistochemical markers. Highgrade ovarian carcinomas (serous and endometrioid) should be tested for BRCA mutation and in case of a detectable tumor mutation, the patient should be referred to an oncogenetic consultation.

Coping with formalin banning in pathology: under vacuum long-term tissue storage with no added formalin

(Ref. SH-047)

Histochemistry and Cell Biology
Luca Mastracci et al.
Read abstract…
Formalin is toxic and has recently been classified as carcinogenic leading to a proposed European formalin ban. But, the pathology use of formalin has however been completely overlooked, and this is proving to be a relevant issue, as no alternative, reliable, tissue fixative is available. Various systems have been proposed to reduce formalin use and exposure; long-term storage and disposal of formalin is also a problem. With this in mind, under vacuum sealing (UVS) systems have been proposed for transportation/storage, however, for how long tissue retains its characteristics (morphological and molecular) is unknown. This study aims to compare histology specimens stored by formalin immersion (FI) and specimens stored after fixation with UVS technique with no additional formalin, at different time periods. Twenty tissue samples (10FI; 10UVS) were stored for different time periods (15 days, 1-2-3-6-12 months) for a total of 120 samples, compared with regard to their morphology, histochemistry, immunoreactivity (24 specific antibodies) and DNA status. All samples showed well-preserved morphology and overlapping staining quality. A significant reduction in immunoreactivity was however identified in the various time periods, particularly for heat pre-treated nuclear antigens, and this commenced earlier (1 month) for FI. UVS storage showed higher DNA content than FI but slightly poorer DNA integrity. These results add important knowledge to the use of UVS in daily practice, as long-term storage of pre-fixed tissue in UVS is not detrimental to the quality of tissue while having the boon of using very little formalin with less operator exposure and lower disposal costs.

Formalin-free surgical specimen management: transforming the workflow

(Ref. SH-048)

Pathology in practice
Cwm Taf Morgannwg UHB, Wales
Read abstract…
An innovative vacuum packing solution has been adopted in order to reduce formalin use and exposure throughout the Cwm Taf Morgannwg University Health Board in Wales, as the following overview of progress illustrates.

Tissues under-vacuum to overcome suboptimal preservation

(Ref. SH-049)

New Biotechnology
Laura Annaratone et al.
Read abstract…
The accuracy of histopathological diagnosis is strictly reliant on adequate tissue preservation, which is completely dependent on pre-analytical variables. Among these variables, the time interval between the end of surgical excision to the onset of fixation (the cold ischemia time) may adversely affect preservation of tissue morphology, influencing the interpretation and reproducibility of diagnosis. During this time interval, the activation of enzymes may produce autolysis and degradation of antigens and nucleic acids, thus potentially affecting immunocytochemical and molecular results. Several studies have described under-vacuum at 4?°C storage of fresh surgical specimens as a safe and reliable method to control cold ischemia and preserve fresh tissues, as well as to standardize fixation times and implement tissue-banking. This review article gives a systematic overview of the advantages and drawbacks of the use of under-vacuum tissue preservation and cooling in surgical pathology, highlighting the impact this procedure may have on diagnostic and experimental pathology. It also documents our experience acquired within daily practice and national and international projects.

Vacuum intraoperative specimen mammography: a novel technique

(Ref. SH-050)

European Journal of Obstetrics & Gynecology and Reproductive Biology
Maria Grazia Baù et al.
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Objective Intraoperative specimen mammography (ISM) is a diffuse technique that allows surgeons to check specimens immediately after lumpectomy. Although the specimen is slightly compressed, the radiological image can be distorted by tissue overlap, and this may affect the evaluation of tumour borders, resulting in extension of the lumpectomy. As ISM may be less precise due to inadequate compression, a vacuum effect was applied to the specimen to increase the precision of margin detection. Study design This study was conducted at St. Anna Hospital Breast Unit, Turin, Italy. Women who underwent lumpectomy for cancer were eligible for inclusion. Both standard ISM (sISM) and vacuum ISM (vISM) were performed. Eighteen specimens obtained after lumpectomy from 1 April 2018 to 31 April 2018 were scanned. sISM (two orthogonal projections) was performed. Next, the specimen was placed in a vacuum, and vISM was performed. The examination was completed with a second orthogonal projection after removal of the vacuum, replacement of the specimen and repositioning of the vacuum. Additional tissue was removed if the surgeon considered that excision was inadequate. Finally, the specimen was sent for definitive histopathological analysis, which is the gold standard for the assessment of surgical margins. Intraoperative histological margin assessment was not performed. The sISM and vISM images and final histopathology reports were compared. Results For sISM, specificity was 47 % [95 % confidence interval (CI) 25–70], sensitivity was 67 % (95 % CI 21–94), positive predictive value (PPV) was 20 % (95 % CI 6–51) and negative predictive value (NPV) was 88 % (95 % CI 53–98). For vISM, specificity was 100 % (95 % CI 80–100), sensitivity was 67 % (95 % CI 21–94), PPV was 100 % (95 % CI 34–100) and NPV was 94 % (95 % CI 72–99). Conclusion These data suggest that the vacuum technique is feasible, cost-saving and yields results that are similar to those from frozen sections but without the limitations, such as prolonged operating time, high variability in sensitivity due to pathologists’ abilities, risk of compromising the histological report, and unreliability for small lumps and ductal carcinoma in situ.

Molecular in vitro diagnostic examinations – Specifications for preexamination process for formalin-fixed and paraffin-embedded (FFPE) tissue

(Ref. SH-051)

European Standard – EVS-EN ISO 20166-4:2021
ISO Directives 2021
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Main points extracted pari passu from the English text version

Advances in surgical specimen management across North Wales

(Ref. SH-052)

Pathology in practice
Betsi Cadwaladr University Health Board
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In order to optimise and standardise the handling of surgical specimens, Betsi Cadwaladr University Health Board has adopted an innovative vacuum-packing solution across its three main hospital sites in North Wales, reducing formalin use, improving quality and cutting costs.