Small Tissue Fragments Research Articles

From worm to germ: Germ cell development and regeneration in planarians.

The specification and proper differentiation of germ cells ensure the propagation of sexually reproducing species. Studies of a wide range of organisms have uncovered several important, conserved features of germ cell development, including the critical roles played by localized niches and somatically derived systemic cues. The planarian Schmidtea mediterranea is an excellent model to study fundamental aspects of germ cell development. Planarians are well known for their remarkable regenerative abilities and can regenerate whole animals from small tissue fragments. This amazing ability is bestowed by neoblasts, pluripotent somatic stem cells that are maintained throughout the planarian's lifetime. Advances in functional genomic methodologies have made planarians a powerful model to investigate the molecular mechanisms underlying germ cell development and reproductive maturation. Here we review recent studies that have led to the discovery of several germ cell-intrinsic factors and somatically derived extrinsic signals important for regulating various aspects of germ cell development. In addition to revealing deep conservation of mechanisms that intrinsically regulate germ cells, these studies also uncover an important function for neuroendocrine control of planarian reproduction as well as novel roles for GPCR signaling in the somatic gonadal niche.

Prostate Osteoblast-Like Cells: A Reliable Prognostic Marker of Bone Metastasis in Prostate Cancer Patients.

The main aim of this study was to investigate the putative association among the presence of prostate cancer cells, defined as prostate osteoblast-like cells (POLCs), and showing the expression of typical morphological and molecular characteristics of osteoblasts, the development of bone metastasis within 5 years of diagnosis, and the uptake of 18F-choline evaluated by PET/CT analysis. To this end, prostate biopsies (n = 110) were collected comprising 44 benign lesions and 66 malignant lesions. Malignant lesions were further subdivided into two groups: biopsies from patients that had clinical evidence of bone metastasis (BM+, n = 23) and biopsies from patients that did not have clinical evidence of bone metastasis within 5 years (BM−, n = 43). Paraffin serial sections were obtained from each specimen to perform histological classifications and immunohistochemical (IHC) analysis. Small fragments of tissue were used to perform ultrastructural and microanalytical investigations. IHC demonstrated the expression of markers of epithelial-to-mesenchymal transition (VIM), bone mineralization, and osteoblastic differentiation (BMP-2, PTX-3, RUNX2, RANKL, and VDR) in prostate lesions characterized by the presence of calcium-phosphate microcalcifications and high metastatic potential. Ultrastructural studies revealed the presence of prostate cancer cells with osteoblast phenotype close to microcalcifications. Noteworthy, PET/CT analysis showed higher uptake of 18F-choline in BM+ lesions with high positivity (≥300/500 cells) for RUNX2 and/or RANKL immunostaining. Although these data require further investigations about the molecular mechanisms of POLCs generation and role in bone metastasis, our study can open new and interesting prospective in the management of prostate cancer patients. The presence of POLCs along with prostate microcalcifications may become negative prognostic markers of the occurrence of bone metastases.

Autotransplantation of fragmented ovarian cortical tissue: a laparoscopic demonstration

To describe and demonstrate a simple and secure procedure for laparoscopic autotransplantation of fragmented ovarian cortical tissue in women with diminished ovarian reserve as part of invitro activation (IVA) of ovarian follicles. Step-by-step video explanation of the surgical procedure with still pictures and surgical video clips to demonstrate the detailed technique. Fertility clinic and obstetrics and gynecology department at a university hospital. Women with idiopathic diminished ovarian reserve and indication for invitro fertilization (IVF), aged 25 to 39years with an antral follicle count bilaterally of ≤5, antimüllerian hormone level of ≤5 pmol/L, and two ovaries. The laparoscopic autotransplantation consists of six steps: [1] obtaining ovarian cortical biopsy samples, [2] preparing the peritoneal pocket, [3] fragmenting the ovarian cortical tissue into pieces of approximately 1mm3, [4] installing the tissue fragments into a catheter, [5] transplanting the tissue fragments into the peritoneal pocket, and [6] closing the peritoneal pocket with a surgical clip. After the procedure, the patients are evaluated with blood samples and ultrasound scans followed by controlled ovarian stimulation. Ethics committee approval was obtained. Feasibility of a six-step laparoscopic autotransplantation procedure using fragmented ovarian cortical tissue. A simple, fast laparoscopic procedure for taking biopsy samples and autotransplanting cortical tissue fragments in an all-in-one procedure ensures the rapid handling and correct placement of the small tissue fragments. The procedure is performed in an outpatient setting with an operation time of 1hour. We have performed this procedure on 20 patients with no complications. Invitro activation is a new, developing option for women in fertility treatment who have diminished ovarian reserve. Fragmentation of murine ovarian tissue has shown to suppress the Hippo pathway, thereby initiating proliferation and growth. This surgical procedure resembles that used when transplanting pieces of frozen-thawed ovarian tissue for fertility restoration, but the fragmented ovarian tissue is only 1mm3, which makes it difficult to transplant. Until now no surgical procedures for transplanting small IVA fragments of cortical tissue has been published. With this video we report in detail a simple way of autotransplanting small fragments of IVA cortical tissue using what is already accessible in the operating theater. Among the many advantages of this procedure are its short duration (1hour) and outpatient setting, which enable fast recovery and minimal postoperative pain. The procedure also allows fast handling and minimal manipulation of the tissue (limited to the fragmentation). The effect of autotransplantation of fragmented tissue in women with diminished ovarian reserve is currently being studied in ongoing trials. If the technique is combined with chemical IVA, a better outcome may be seen.

Confocal Fluorescence Microscopy Platform Suitable for Rapid Evaluation of Small Fragments of Tissue in Surgical Pathology Practice.

Rapid advances in the fields of biophotonics, computer science, and instrumentation have allowed for high-resolution imaging of biologic tissues. To evaluate the quality of images from an optimized confocal fluorescence microscopy (CFM) platform for rapid evaluation of small fragments of tissue, compared with hematoxylin-eosin staining. Tissue fragments (up to 1.0 × 0.3 cm) were stained with 0.6 mM acridine orange for 60 seconds and imaged using a CFM platform at 488-nm and 785-nm wavelength. The imaged tissues were then fixed in formalin and processed to generate hematoxylin-eosin-stained tissue sections. The quality of CFM images was scored on a scale of 0 to 3 on the basis of the percentage of the CFM images with recognizable tissue architecture (0, 0%; 1, <20%; 2, 20%-50%; 3, >50%). The diagnoses made using CFM images were compared with those made using histopathologic analysis of the hematoxylin-eosin-stained tissue sections. We imaged 118 tissue fragments obtained from 40 breast, 23 lung, 39 kidney, and 16 liver surgical excision specimens. We acquired CFM images in 2 to 3 minutes; 95.8% (113 of 118) of images showed a quality score of 3, and 4.2% (5 of 118) had a score of 2. We achieved a sensitivity of 95.5%, specificity of 97.3%, positive predictive value of 95.5%, and negative predictive value of 97.3%. Our results demonstrate the suitability of the CFM platform for rapid and accurate evaluation of small tissue fragments in surgical pathology practice.

First Report of Leaf Spot of Spinach Caused by Stemphylium beticola in Italy.

During the spring and autumn of 2017, 40-day-old spinach (Spinacia oleracea L.) plants, cultivar Crocodile, grown in a commercial field in the Asti province in Piedmont (Northern Italy), showed symptoms of a previously unknown foliar disease. The first symptoms developed at air temperatures ranging from 10 to 22°C. Five to 25% of plants showed small circular, yellow to pale brown leaf spots (1 to 2 mm in diameter), with a well-defined border, generally surrounded with a yellow halo. As the lesions expanded, they changed from circular to elliptical or irregular, becoming necrotic and broken in the center. Small tissue fragments were excised from symptomatic leaves, dipped in a solution containing 1% sodium hypochlorite for 5 s, washed in sterile water, dried on sterile paper, and plated on potato dextrose agar. After 7 days of incubation under 12-h fluorescent light at 22°C, 80% of fragments were colonized by the same fungus. Examination of the isolates IT27, IT28, and IT50 grown on V8 agar medium (200 ml of V8, 15 g of agar, 0.5 g of CaCO₃, and 1 liter of distilled water) revealed multicellular, light brown pigmented, obclavate or subspherical conidia, measuring 19.2 to 31.9 μm long × 10.7 to 21.4 μm wide (length/width ratio 1.1 to 1.4), with two to four transverse and three to four longitudinal septa. Conidia developed on a distinctive brown conidiophore with a terminal enlargement. These morphological characters permitted identifying the fungus as Stemphylium sp. (Simmons 1969). Genomic DNA from 7-day-old pure cultures of IT27, IT28, and IT50 were extracted with E.Z.N.A. Plant DNA Kit (Omega Bio-Tek). Polymerase chain reaction (PCR) was performed using primers ITS1/ITS4 to amplify the internal transcribed spacer, the intergenic region between 28 S and 18 S sequences of the ribosomal RNA, including the 5.8 S sequence. The PCR products were purified and sent for sequencing to BMR Genomics (Padova, Italy). BLASTn analysis (Altschul et al. 1997) of the three isolates (483 bp) showed 100% similarity with Stemphylium beticola CBS 141024 (GenBank no. NR_154925.1). Additionally, alignment with the CBS isolates used by Woudenberg et al. (2017) and the isolates from spinach was carried out using Clustal W with Mega 6.0.6 (Tamura et al. 2013). Sequence analysis of the same length sequences was performed using the maximum likelihood method with the Kimura two-parameter model plus gamma distribution. The sequences from IT27, IT28, and IT50 isolates clustered with the S. beticola strains CBS 141024, CBS 141025, and CBS 141026 (GenBank nos. KU850520, KU850521, and KU850522, respectively), allowing confirmation of the identity of isolates from spinach as S. beticola. The sequences from the isolates IT50, IT27, and IT28 were deposited in GenBank with the accession numbers MG913379, MG913380, and MG91338, respectively. The pathogenicity test was carried out by spraying a conidial suspension of 10⁵conidia/ml from a single-spore culture of strain IT50 onto leaves of 30-day-old spinach cultivar Crocodile plants, maintained under >90% relative humidity conditions for 5 days after inoculation. Pots (five per treatment, 10 plants/pot) were kept in a growth chamber at an average temperature of 20°C. The first lesions developed on leaves 8 days after inoculation, whereas control plants remained healthy. The pathogenicity test was conducted three times, providing the same results. Based on morphological and molecular analysis, the same fungus was consistently reisolated from the lesions. This is to our knowledge the first report of S. beticola on spinach in Italy and probably in Europe (Farr and Rossman 2018), although the same species has been reported on spinach in the United States (Woudenberg et al. 2017). The importance of the disease is at present limited but could rapidly increase in the cultivation areas where spinach is intensively grown.

Vascular Morphogenesis in the Context of Inflammation: Self-Organization in a Fibrin-Based 3D Culture System.

Introduction: New vessel formation requires a continuous and tightly regulated interplay between endothelial cells with cells of the perivascular microenvironment supported by mechanic-physical and chemical cues from the extracellular matrix.Aim: Here we investigated the potential of small fragments of synovial tissue to form de novo vascular structures in the context of inflammation within three dimensional (3D) fibrin-based matrices in vitro, and assessed the contribution of mesenchymal stromal cell (MSC)-immune cell cross-talk to neovascularization considering paracrine signals in a fibrin-based co-culture model.Material and Methods: Synovial tissue fragments from patients with rheumatoid arthritis (RA) and inflammatory osteoarthritis (OA) were cultivated within 3D fibrin matrices for up to 4 weeks. Cellular and structural re-arrangement of the initially acellular matrix were documented by phase contrast microscopy and characterized by confocal laser-scanning microscopy of topographically intact 3D cultures and by immunohistochemistry. MSC-peripheral blood mononuclear cell (PBMC) co-cultures in the 3D fibrin system specifically addressed the influence of perivascular cell interactions to neo-vessel formation in a pro-inflammatory microenvironment. Cytokine levels in the supernatants of cultured explant tissues and co-cultures were evaluated by the Bio-Plex cytokine assay and ELISA.Results: Vascular outgrowth from the embedded tissue into the fibrin matrix was preceded by leukocyte egress from the tissue fragments. Neo-vessels originating from both the embedded sample and from clusters locally formed by emigrated mononuclear cells were consistently associated with CD45+ leukocytes. MSC and PBMC in co-culture formed vasculogenic clusters. Clusters and cells with endothelial phenotype emerging from them, were surrounded by a collagen IV scaffold. No vascular structures were observed in control 3D monocultures of PBMC or MSC. Paracrine signals released by cultured OA tissue fragments corresponded with elevated levels of granulocyte-colony stimulating factor, vascular endothelial growth factor and interleukin-6 secreted by MSC-PBMC co-cultures.Conclusion: Our results show that synovial tissue fragments with immune cell infiltrates have the potential to form new vessels in initially avascular 3D fibrin-based matrices. Cross-talk and cluster formation of MSC with immune cells within the 3D fibrin environment through self-organization and secretion of pro-angiogenic paracrine factors can support neo-vessel growth.

Characterization of artificial tumor‐derived monolayers from long‐term MCF‐7 breast adenocarcinoma constructs using flow cytometry and immunocytological analysis.

For the past decade our laboratory has been engaged in developing and testing a variety of 3D artificial tissues, both normal and pathologic, using several types of natural de‐cellularized extracellular matrix materials. Throughout this work, one notable observation stood out with all of the tissues generated, specifically, 3D constructs transitioned through a series of “developmental” stages reflective of in‐vivo tissue development. These stages involve the early colonization and establishment of the basic tissue structure followed by a period of maturation and a final relatively long term stable mature tissue. In this case, we employed de‐cellularized and de‐calcified bovine trabicular bone matrix and MCF‐7 cells to develop artificial tumor tissues which were maintained for periods of 1, 2, 3 and 4 years respectively. During these culture periods, each tissue displayed the stages listed above and following the initial colonization period of one to two weeks each also began to shed cells and later spheres which, in turn, established monolayers of “post‐scaffold” cells. These monolayers were mainly established as multiple individual colonies by shed and explanted spheres or small tissue fragments. These various colonies eventually coalesced to form a single monolayer. Flow cytometric analysis of monolayers from each of the four culture periods demonstrate enhanced populations of cells with immunophenotypes for breast cancer stem cells based on CD44/CD24 labeling. In addition, individual cellular morphology as well as culture growth patterns were consistently changed from the baseline standard MCF‐7 cultures. These cells also displayed a substantially reduced cell‐cell adhesion pattern leading to loose patches in place of the relatively tight colonies seen with original MCF‐7 cultures. Taken together, these cellular modifications resulting from long‐term culture of artificial tissues support our interpretation that such constructs reflect the process of tumor progression in‐vivo and result in enhanced metastatic cell populations.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impregnation as a method for preservation of relics used by the Christian Church

Relics are a special type of human body remains, which are venerated by the faithful due to the prominence the person had achieved during lifetime. Even if no such claims for incorruptibility are made, body relics have to be preserved in some fashion, in order to allow for their long-term viewing and veneration. The present report summarizes the available data regarding the impregnation as a method for body preservation, used by the Christian Church to conserve body relics. Impregnation is a widely used method, both by the Orthodox as well as by the Roman Catholic Church. It is known that bodies are treated with different protective mixtures on variable intervals after death. Public worship historically involved pouring of oil over relics. Moreover, small tissue fragments are embedded in wax blocks or are sealed with a mixture of molten wax and other resins. All of those methods are in fact impregnation with compounds with high molecular mass as a final stage of a preservation procedure. This impregnation of a tissue already reached some degree of dehydration or other preservation ensures longevity and protection of the relics by greatly slowing down the natural processes of decomposition and disintegration.

First Report of Bacterial Leaf Blight of Tea (Camellia sinensis) Caused by Acidovorax avenae in China

Tea (Camellia sinensis L.) is an economically important crop widely cultivated for leaves in China. Leaf and young shoot blights were observed on some commercial gardens of tea (Camellia sinensis, cv. Yinghong IX) in Guangzhou in April from 2013 to 2015, China. The disease occurred initially on the young leaves producing water-soaked spots. When the disease further developed, the spots merged together into large patches and killed the leaf blade. The symptoms developed on the young shoots about two weeks later. Disease incidences ranged from 20 to 30% in the affected fields. Signs of bacterial streaming from all infected leaves were observed under microscope, and no fungal mycelium or spore was found from the diseased areas. Bacterial isolates were obtained by surface-sterilizing small fragments (5 × 5 mm) of symptomatic leaf tissues in 0.5% NaOCl, rinsing the sections in sterilized water twice, and streaking on Luria-Bertani (LB) plates. Bacterial colonies were convex, beige-tan colored, round and non-mu...

Multiplexed analysis of fixed tissue RNA using Ligation in situ Hybridization.

Clinical tissues are prepared for histological analysis and long-term storage via formalin fixation and paraffin embedding (FFPE). The FFPE process results in fragmentation and chemical modification of RNA, rendering it less suitable for analysis by techniques that rely on reverse transcription (RT) such as RT-qPCR and RNA-Seq. Here we describe a broadly applicable technique called ‘Ligation in situ Hybridization’ (‘LISH’), which is an alternative methodology for the analysis of FFPE RNA. LISH utilizes the T4 RNA Ligase 2 to efficiently join adjacent chimeric RNA–DNA probe pairs hybridized in situ on fixed RNA target sequences. Subsequent treatment with RNase H releases RNA-templated ligation products into solution for downstream analysis. We demonstrate several unique advantages of LISH-based assays using patient-derived FFPE tissue. These include >100-plex capability, compatibility with common histochemical stains and suitability for analysis of decade-old materials and exceedingly small microdissected tissue fragments. High-throughput DNA sequencing modalities, including single molecule sequencing, can be used to analyze ligation products from complex panels of LISH probes (‘LISH-seq’), which can be amplified efficiently and with negligible bias. LISH analysis of FFPE RNA is a novel methodology with broad applications that range from multiplexed gene expression analysis to the sensitive detection of infectious organisms.

Comparative study of cell block versus centrifuged smear examination from aspirates of cystic lesions

Background: Cystic fluids encountered during routine FNA poses a diagnostic challenge to cytopathologists due to its low cell yield with high dispersal of cells on conventional centrifuged smears (CS). Cell Block (CB) technique enables retrieval of small tissue fragments from fluids, thereby providing scope for better morphology and material for ancillary techniques which help in improving the diagnosis. Aim : To compare the efficacy of CB versus CS, in cyto-diagnosis of cystic lesions. Methods : This observational study was conducted on a total of 50 fluid samples aspirated from cystic lesions during routine FNA and fluids aspirated peri-operatively from cystic ovarian lesions. Divided into two equal parts, one part was processed for CS and CB by Fixed Sediment Method and relevant immunohistochemistry was performed. CSs were categorized as positive for malignancy, benign diagnosis, Inadequate for opinion and suspicious for malignancy. CBs were categorized as; no material, Non-contributory (CS+, CB-), confirms the smear diagnosis and establishes a specific diagnosis. The comparison between CS and CB was analysed by Chi- square test & kappa test. Results: Out of the 50 cases, 35(70%) were given a benign diagnosis, 10 (20%) were positive for malignancy, 2(4%) were suspicious and 3(6%) were inadequate for opinion on CS. In CB out of 50 cases, 29 of them confirmed/established a diagnosis and 21 cases were non diagnostic / non-contributory. CB gave an improved diagnosis in 2 out of 10 (20% ) malignant cases and 2 out of 35 (5.7%) benign cases.( p value = 0.00054, Kappa value =0.34) Conclusions: CBs complemented CS, more so in malignant lesions by preserved architecture. Aspirates from multiple sites of the cystic lesions (with/without radiological assistance) pooled as one specimen yielded better material for CBs and ancillary techniques like histochemistry and IHC. DOI: 10.21276/APALM.1249

Plant materials used as temper in the oldest Neolithic pottery from south-eastern Poland

Plant materials were frequently used as a temper as an important part of the process of making pottery. However, identification of the presence of tempering material and its taxonomic composition are still uncommon practices. This paper presents the results of a study of plant remains observed in pottery dated to the oldest Neolithic from south-eastern Poland, as a tool for detecting or confirming changes in the method of making the pottery. In previous studies, plant material was noticed sporadically in coarse ware and its identification was based on macroscopic plant morphology. On the other hand, remains of plants were not usually observed in the other finer ware types since their surfaces were frequently smoothed and decorated, while their sections were very thin. Therefore, in the present study, these groups of pottery have been studied in detail. A preliminary observation of surfaces and fresh sections of selected potsherds has shown that plant temper was neither evident nor abundant in them. However, with microscopy, small fragments of plant tissues and their imprints have been seen inside the clay. Their identification was not possible on the basis of plant morphology, but was carried out with the help of plant anatomy, especially by observing the microscopic features of plant epidermis. Previous archaeobotanical studies from the area indicated that the plants used as temper in coarse ware mostly included remnants of cereal chaff. Consequently, this paper will test this hypothesis in the case of the finer Neolithic ceramics.

Cell and Tissue Display: An Alternative Multipurpose Tool for Microscopy.

We developed a method, termed Cell and Tissue Display (CTD), for embedding 16 or more different tissue samples in multi-compartment agarose blocks. The CTD-generated blocks allow uniform multiplexing of cell lines and small tissue fragments within a single histologic block. The distribution of individual cells within the CTD blocks is improved, likely because the individual agarose compartments are small and uniform. The composition of each CTD block can be customized based on intended use. Some potential uses of CTD histologic blocks include improved sectioning of small tissue fragments, such as needle biopsy specimens or punch biopsies; multiplexing of tissue fragments within a single block; and the generation of control slides for laboratory proficiency testing. .

Chondroblastic osteosarcoma: Cytomorphologic characteristics and differential diagnosis on FNA.

Chondroblastic osteosarcoma (COS) is a uniformly fatal bone malignancy if not diagnosed and treated appropriately in a timely manner. Fine-needle aspiration (FNA) of osseous lesions is routinely performed in major medical centers. Appropriate characterization of the tumor will significantly influence patient management and outcomes. A retrospective review of the cytopathology archives of 2 large tertiary care centers for a 15-year period (2001-2015) revealed 17 cases of COS (9 primary, 6 recurrent, and 2 metastatic cases) in 16 patients. Clinical outcome and histopathologic follow-up were reviewed and correlated. There were 9 male and 7 female patients (male-to-female ratio of approximately 1:1), ranging in age from 12 to 70 years (mean age, 29.2 years). The classic locations for osteosarcoma were commonly involved, such as the long bones around the region of the knee in 4 cases and the proximal humerus in 1 case. However, other "nonclassic" sites also were noted to be commonly involved, including the distal tibia or fibula (4 cases), sacroiliac region (4 cases), mandible (3 cases), and skull (1 case). Cytomorphologic characteristics were high cellularity; predominantly discohesive, single cells; small tissue fragments; background osteoid and chondroid matrix; spindled cells and plasmacytoid cells with moderate to abundant basophilic vacuolated cytoplasm and occasional multiple cytoplasmic processes; round to oval nuclei with anisonucleosis; and small to prominent nucleoli. There were scattered binucleated and multinucleated osteoclast-like giant cells. Cases with high-grade morphology demonstrated marked pleomorphism and abundant mitoses. The differential diagnosis of COS includes chondroblastoma, chondrosarcoma, and chondroid chordoma. A definitive diagnosis can be made with clinical and radiological correlation. Cancer Cytopathol 2016;124:493-500. © 2016 American Cancer Society.

Rapid DNA analysis for automated processing and interpretation of low DNA content samples.

BackgroundShort tandem repeat (STR) analysis of casework samples with low DNA content include those resulting from the transfer of epithelial cells from the skin to an object (e.g., cells on a water bottle, or brim of a cap), blood spatter stains, and small bone and tissue fragments. Low DNA content (LDC) samples are important in a wide range of settings, including disaster response teams to assist in victim identification and family reunification, military operations to identify friend or foe, criminal forensics to identify suspects and exonerate the innocent, and medical examiner and coroner offices to identify missing persons. Processing LDC samples requires experienced laboratory personnel, isolated workstations, and sophisticated equipment, requires transport time, and involves complex procedures. We present a rapid DNA analysis system designed specifically to generate STR profiles from LDC samples in field-forward settings by non-technical operators. By performing STR in the field, close to the site of collection, rapid DNA analysis has the potential to increase throughput and to provide actionable information in real time.ResultsA Low DNA Content BioChipSet (LDC BCS) was developed and manufactured by injection molding. It was designed to function in the fully integrated Accelerated Nuclear DNA Equipment (ANDE) instrument previously designed for analysis of buccal swab and other high DNA content samples (Investigative Genet. 4(1):1–15, 2013). The LDC BCS performs efficient DNA purification followed by microfluidic ultrafiltration of the purified DNA, maximizing the quantity of DNA available for subsequent amplification and electrophoretic separation and detection of amplified fragments. The system demonstrates accuracy, precision, resolution, signal strength, and peak height ratios appropriate for casework analysis.ConclusionsThe LDC rapid DNA analysis system is effective for the generation of STR profiles from a wide range of sample types. The technology broadens the range of sample types that can be processed and minimizes the time between sample collection, sample processing and analysis, and generation of actionable intelligence. The fully integrated Expert System is capable of interpreting a wide range or sample types and input DNA quantities, allowing samples to be processed and interpreted without a technical operator.

Clonality Testing in Veterinary Medicine

The accurate distinction of reactive and neoplastic lymphoid proliferations can present challenges. Given the different prognoses and treatment strategies, a correct diagnosis is crucial. Molecular clonality assays assess rearranged lymphocyte antigen receptor gene diversity and can help differentiate reactive from neoplastic lymphoid proliferations. Molecular clonality assays are commonly used to assess atypical, mixed, or mature lymphoid proliferations; small tissue fragments that lack architecture; and fluid samples. In addition, clonality testing can be utilized to track neoplastic clones over time or across anatomic sites. Molecular clonality assays are not stand-alone tests but useful adjuncts that follow clinical, morphologic, and immunophenotypic assessment. Even though clonality testing provides valuable information in a variety of situations, the complexities and pitfalls of this method, as well as its dependency on the experience of the interpreter, are often understated. In addition, a lack of standardized terminology, laboratory practices, and interpretational guidelines hinders the reproducibility of clonality testing across laboratories in veterinary medicine. The objectives of this review are twofold. First, the review is intended to familiarize the diagnostic pathologist or interested clinician with the concepts, potential pitfalls, and limitations of clonality testing. Second, the review strives to provide a basis for future harmonization of clonality testing in veterinary medicine by providing diagnostic guidelines.

The sooner the better: Our laboratory experience with chorionic villus sampling (CVS) HLA typing prior to HSCT

Aim Allogeneic Hematopoietic stem cell transplantation(HSCT) will prolong and improve the quality of life of babies born affected by inherited metabolic diseases. Genetic testing and preparation for a potential HSCT starts at 10 weeks of mother’s pregnancy. This study aims to describe our strategy to obtain an HLA typing from the minuscule tissue fragments submitted for HLA typing in preparation for an HSCT just after birth. Methods Mothers carrying babies that may be affected with Hurler or Krabbe A syndromes are the most frequent challenges at Duke Hospital. As needed, CVS is submitted to our laboratory and DNA is extracted using a micro method developed specifically for these samples to allow High Resolution HLA typing. Since maternal blood contamination of the CVS is a common finding, High Resolution HLA typing on the parents is also done to assist with the identification of all HLA alleles present on the CVS. Our laboratory sequencing strategy for HLA-A, B, C, DRB1 & DQB1 identifies most alleles in a hemizygous form and duplication of the same allele (potential homozygosis) is always resolved by both parents HLA typing. All positive SBT amplification reactions obtained for the CVS DNA are sequenced, and based on parents typing results we are able to identify, first the paternal haplotype present followed by the maternal one. The software SeqPilot®, used for high resolution data analysis, allows the analysis of all positive reactions chosen to be sequenced. Results The CVS samples we have tested (total of 20 cases) have provided the correct pool of alleles expected to be present and it facilitated cord blood searches for these unborn babies, providing an increased chance for successful treatment. Conclusions The HLA typing strategy we apply to CVS is only possible due to our level of expertise extracting DNA from small tissue fragments and the flexibility and data quality offered by the High Resolution HLA Typing methodology and software data analysis we use. The affected and genetic carrier unborn babies get a much higher chance at cure and survival when their diagnosis and treatment can be done earlier in their lives.

Imaging and identification of endogenous peptides from rat pituitary embedded in egg yolk.

Mass spectrometry imaging (MSI) can provide accurate data containing the spatial distribution of endogenous peptides in tissue sections without previous treatment. One of the key issues in analyzing small samples is establishing a proper technique for mounting and manipulating collected tissue in order to avoid contamination of the sample with optimal cutting temperature (OCT) resin. We present a method for embedding rat pituitary tissue in a frozen egg yolk block, which enables its further imaging in experiments on a matrix-assisted laser desorption/ionization (MALDI) mass spectrometer with time-of-flight (TOF) analyzer. Embedding the sample in the egg yolk prevents contamination from the OCT resin, which decreases MALDI signal quality. In the present study we detected numerous m/z peaks related to endogenous peptides. We identified fifteen peptides and their post-translational modifications by tandem mass spectrometry (MS/MS) directly on tissue sections of the hypophysis posterior and intermediate lobes; among these peptides were vasopressin, oxytocin, copeptin, melanocyte-stimulating hormones and beta-endorphin. We also showed that egg yolk itself does not affect localization of peptides in the pituitary. Egg yolk embedding enables preparation of tissue sections from small tissue fragments to organs such as the pituitary gland, which is suitable for localization and identification of endogenous peptides by the MALDI-MSI and MALDI-MS/MS techniques.

Rib Shaft Tuberculous Granuloma an Osteolytic Malignant Mimic Lesion: A Case Report

SESSION TITLE: Infectious Disease Global Case Reports SESSION TYPE: Global Case Report PRESENTED ON: Tuesday, October 28, 2014 at 01:30 PM 02:30 PM INTRODUCTION: Chest wall involvement is a rare manifestation of tuberculosis.Globally, musculoskeletal tuberculosis (TB) occurs in 1-3% of patients with TB, while TB of the chest wall constitutes 1% to 5% of all cases of musculoskeletal TB.The risk of medical laboratory staff acquiring TB is 5 times more than non laboratory workers. CASE PRESENTATION: 36 year-old male was presented with back pain and non-painful swelling on the left side of the chest anterior over a period of 3 months. The medical history of this patient was unremarkable. He had no pulmonary symptoms, such as a productive cough, or other symptoms.General examination revealed that the patient was a febrile and well nourished.A large, firm, non-mobile, and non-tender mass in the left anterior thoracic wall , overlying skin appeared normal, with no wounds, scars, rash, or sinuses. no other swellings in the body, no lymph nodes enlargement. A plain radiograph of the chest revealed normal findings. CT showed : osteolytic lesion with swelling in the left 7Th rib anterior arc, suggesting malignancy. CT also shows destruction of the rib shaft with some depress of bone in the side of the soft tissue swelling with segment of about 2 cm break in the continuity of the rib. The white blood cell count was normal. All biochemical examinations as well as erythrocyte sedimentation rate were within normal ranges. A tuberculin test was positive 15 mm. HIV, HCV and HBS Ag tests were negative. CT guided fine needle aspiration was done and revealed small fragments of chronic granulomatous inflammatory tissue in a background of traumatic blood fragments of fibrocollagenous tissue contains some microgranulomas.There were composed of loose aggregates of epithelioid cells and dense aggregates of lymphocytes around. focal areas of early caseation necrosis.No evidence of neoplasia was seen.The patient is currently receiving daily antituberculous chemotherapy. DISCUSSION: The granulomatous inflammation of tuberculosis commonly affects the lungs and the hilar lymph nodes, rarely to affect the chest wall and rarer to cause TB granulomatous osteolytic mass in the rib shaft. Chondroma, osteochondroma, fibrous dysplasia, and lipoid granuloma are the common benign tumors involving the ribs while the malignant lesions are chondrosarcoma, myeloma multiplex and secondary deposits from the lung and breast. All these present as expansion of the rib at the affected site, lytic lesion or pathological fractures. Therefore, this case of osteolytic rib shaft mass with bone debris inside without pulmonary, pleural affection and moreover no central caseation or history of tuberculosis or toxemic manifestation make the case more mimic tumor in the rib.Radiologically, presence of an osteolytic lesion, widening of the rib with periosteal reaction and presence of sequestrum may indicate tuberculosis. A solitary rib involvement usually present and the most frequent location is the rib shaft (60%) (1) and this was evident in our case. In addition to history and a good clinical examination, uses of CT-scan and percutaneous needle biopsy CT guided, remain the gold standard for the diagnosis and that was followed in our case. However, Chang et al.(2) could confirm TB in all of their twelve cases only following rib resection, and opinionated that surgery is a final diagnostic and therapeutic option in parietal chest wall TB. FREE TO VIEW http://journal.publications.chestnet.org/article.aspx?articleID=1913027 1 of 2 5/12/2015 7:02 PM PDF created with pdfFactory trial version www.pdffactory.com Tuberculosis infections have been increasing in incidence during the last decades for a variety of reasons, including increasing numbers of patients with immunity-depressive diseases, drug resistance, aging population, and health care worker exposure (3). In the current case, the patient is medical laboratory technician for more than 14 years. He manipulated TB suspected samples. Also, absence of TB history in close contacts may increase the possibility of laboratory acquired infection CONCLUSIONS: The granulomatous inflammation of tuberculosis may mimic osteolytic lesion in ribs of the thoracic cage especially if early without radiologic evidence of caseation, this can occurs in immunocompetent. Reference #1: Chang JH, Kim SK, Lee WY. Diagnostic issues in tuberculosis of the ribs with a review of 12 urgically proven cases. Respirology 1999 Sep;4:249253. Reference #2: Faure E, Souilamas R, Riquet M, Chehab A, Le Pimpec-Barthes F, Manac’h D, Debesse B. Cold abscess of the chest wall: a surgical entity? Ann Thorac Surg 1998 Oct; 66:1174-1178. Reference #3: Watts HG, Lifeso RM: Current concepts review: tuberculosis of bones and joints. J Bone Joint Surg Am 1996, 78:288-98. DISCLOSURE: The following authors have nothing to disclose: Abdelbaset Saleh, Magda Ahmad, Nabil Awadalla No Product/Research Disclosure Information Copyright © 2015 American College of Chest Physicians http://journal.publications.chestnet.org/article.aspx?articleID=1913027 2 of 2 5/12/2015 7:02 PM PDF created with pdfFactory trial version www.pdffactory.com

Optimal methods for recovery of transplantable stem cells from frozen/thawed human testicular tissue

Spermatogonial stem cell (SSC)-based technologies may have application for preserving and/or restoring male fertility. We evaluated the recovery of stem and progenitor spermatogonia from frozen/thawed testicular cell suspensions versus testicular tissue fragments frozen at a controlled rate (CR) or by vitrification. Laboratory study using human tissue. Human testicular tissue was obtained through the Center for Organ Recovery and Education with approval from the University of Pittsburgh Institutional review board (#0506140). Testicular tissue was digested with enzymes to produce a cell suspension and frozen at CR or minced into large (6-10 mm3) or small (3-5 mm3) testicular tissue fragments and frozen at CR or by vitrification. The efficiency of each technique was analyzed by immunocytochemistry (ICC) for the spermatogonia marker UTF1 and human to nude mouse xenotransplantation. Cryopreservation method had a significant effect on the recovery of UTF1 positive spermatogonia per gram of tissue (p<0.001). Large testicular tissue fragments frozen at CR had the greatest number of UTF1 cells/g tissue (14.9±2.2), followed by CR small (10.4±1.3), vitrified small (5.4±0.9), vitrified large (4.4±0.5), and frozen/thawed cell suspension (1.5±0.2). The difference in UTF1 positive cell recovery from CR large tissue fragments versus CR cell suspension was statistically significant (14.9 vs. 1.5, p=0.027). Human to nude mouse xenotransplants confirmed that recovery of transplantable SSCs (colonies/g tissue) from CR small (1045.9±240.6) and CR large (570.0±147.1) testicular tissue fragments was greater than the CR cell suspension (62.6±12.3, p<0.05). Recovery of transplantable stem cells from vitrified small (562.4±186.0) and vitrified large (75.4±33.2) testicular tissue fragments was not significantly greater than the CR cell suspension (p>0.05). CR freezing of testicular tissue fragments (large or small) leads to the greatest recovery of UTF1 positive spermatogonia and transplantable SSCs. Recovery of SSCs from vitrified small testicular tissue fragments was also very good. Freezing and thawing of intact testicular tissue is compatible with several downstream applications, including testicular tissue grafting, testicular tissue organ culture or digestion with enzymes to produce a cell suspension for SSC transplantation.