Cell count-based triaging of cytology samples for cell block preparation.

OBJECTIVE: Cytology samples can sometimes yield cell blocks of low cellularity that are inadequate for cytomorphological evaluation and ancillary studies. The purpose of this study was to analyse the value of cell count based-triaging of samples for cell block preparation. METHODS: Cell blocks were prepared from different cytology samples using HistoGel™ according to the manufacturer's instructions following cell count assessment. The adequacy of cell blocks was semi-quantitatively scored as empty, scanty, moderate or highly cellular by evaluating corresponding haematoxylin and eosin (H&E) sections. Cell blocks with moderate and highly cellular H&E sections were considered adequate whereas cell blocks with empty and scanty cells in H&E sections were inadequate for cytomorphological evaluation. The average, range and a median number of cells (×106 ) embedded in adequate and inadequate cell blocks were compared. RESULTS: Among 54 cell blocks prepared from fine needle aspiration biopsies (FNAs, 32), effusions (4), brushings (13) and cell lines (5), 20 cell blocks (37%) yielded moderate or highly cellular H&E sections and were considered adequate whereas 34 (63%) cell blocks were empty or scanty and thus considered inadequate. The average (± SD) number of cells (×106 ) embedded in adequate and inadequate cell blocks was 2.1 ± 4.3 and 0.5 ± 0.3, respectively. Samples containing at least 1 × 106 cells yielded adequate cell blocks in 12/16 cases (75%) whereas samples with less than 1 × 106 cells in 13/38 (34%) cases. CONCLUSION: The present study showed that cell count-based triaging of cytology samples could reduce the number of low cellular cell blocks.

Histogel-based techniques for embedding organoids in paraffin blocks enable high throughput downstream histopathological analyses.

Organoids are in vitro tissue models derived from human or animal primary tissues or stem cells that allow for studying three-dimensional (3D) tissue biology, toxicity testing, biomarker evaluation, and assessment of compound efficacy, supplementing or potentially minimizing use of animal models. Organoids are typically cultured in a 3D format within an extracellular matrix and, at the end of an experiment, can be further processed for various cellular or molecular readouts. Analysis often relies on whole mount immunolabeling for markers of interest, which consumes the entire sample/well, thereby limiting sample availability for downstream assays. In addition, 3D cultures become more friable after fixation and are susceptible to sample loss during washing steps. In contrast, by fixing and processing organoids to a paraffin block, dozens or hundreds of unstained slides can be generated, enabling robust characterization via multiple assays, including histologic evaluation and (immuno)histochemical stains, thus maximizing the yield of these time- and labor-intensive cultures. Here we describe three methods to process 3D Matrigel cultures into paraffin blocks using Histogel as an embedding agent. The three techniques all yield high-quality sections but vary in complexity of implementation at different steps, and their application for different use cases is discussed.

Histo-pillar strip for optimal histogel block construction and biomarker analysis in 3D-lung cancer patient-derived organoids.

This study proposed an optimized histogel construction method for histological analysis by applying lung cancer patient-derived organoids (PDOs) to the developed histo-pillar strip. Previously, there is the cultured PDOs damage problem during the histogel construction due to forced detachment of the Matrigel spots from the 96-well plate bottom. To address this issue, we cultured PDO on the proposed Histo-pillar strips and then immersed them in 4% paraformaldehyde fixation solution to self-isolate PDO without damage. The 4µl patient-derived cell (PDC)/Matrigel mixtures were dispensed on the surface of a U-shaped histo-pillar strip, and the PDCs were aggregated by gravity and cultured into PDOs. Cultured PDOs were self-detached by simply immersing them in a paraformaldehyde fixing solution without physical processing, showing about two times higher cell recovery rate than conventional method. In addition, we proposed a method for embedding PDOs under conditions where the histogel temperature was maintained such that the histogel did not harden, thereby improving the problem of damaging the histogel block in the conventional sandwich histogel construction method. We performed histological and genotyping analyses using tumor tissues and PDOs from two patients with lung adenocarcinoma. Therefore, the PDO culture and improved histogel block construction method using the histo-pillar strip proposed in this study can be employed as useful tools for the histological analysis of a limited number of PDCs.

Cell blocks in cytology: review of preparation methods, advantages, and limitations.

Cell blocks are cytologic preparations that are processed as paraffin embedded blocks in a manner comparable to formalin-fixed paraffin-embedded tissue in surgical pathology. In addition to serving as an adjunct to other cytologic preparations for morphologic diagnosis, cell blocks play an increasingly important role as they yield tissue sections that can be utilized for ancillary testing such as immunohistochemical stains and molecular studies. While essentially universally viewed as playing a pivotal role in cytopathology practice, there are various factors that limit their use in practice and contribute to dissatisfaction with cell block quality. Cell block preparation, as opposed to tissue processing in surgical pathology, is more variable with many different protocols in use today. This review explores the most commonly used cell block preparation techniques currently in use with review of the unique advantages and limitations each method presents. The goal of this work is to serve as a resource that can aid in making more informed decisions about which cell block protocol may work best for individual laboratories.

Impact of a Modified HistoGel Method for Processing Endocervical Curettage Specimens on Diagnostic Yield.

OBJECTIVES: Endocervical curettage (ECC) specimens may be limited by scant tissue. We evaluated whether a cellular concentration processing method could improve their diagnostic quality. METHODS: Between October 2018 and June 2019, ECC specimens were assigned chronologically to one of two groups: nonconcentrated ECC (NECC) or concentrated ECC (CECC). NECC specimens underwent routine histologic processing. CECC specimens were processed using a published HistoGel-based cell block method. We reviewed diagnoses for ECCs, concurrent cervical biopsies and/or loop electrosurgical excision procedures (LEEPs), and preceding Papanicolaou (Pap) smears. We performed multivariate logistic regression analyses to evaluate the impact of processing method on ECC adequacy and discordance between Pap smear and worst tissue diagnoses. RESULTS: NECC and CECC adequacy was 88.2% and 84.7% (P = .06). ECC adequacy was greater if concurrent biopsy/LEEP was performed (odds ratio [OR] = 1.76, P < .01). Discordance between Pap smear and worst tissue diagnoses was 9.5% and 13.3% (P = .04) for cases with NECC and CECC processing, although processing method was not significant in multivariate analysis (OR = 0.74, P = .11). Adequate ECC sampling and concurrent biopsy/LEEP were independently associated with concordance between Pap smear and worst tissue diagnosis (OR = 0.46, P < .01 and OR = 0.65, P = .02). CONCLUSIONS: ECC processing method did not significantly affect either specimen adequacy (P = .06) or diagnostic discordance (P = .11) when controlled for other factors.

Cell blocks in veterinary medicine: A comparison of two methods (cell tube and agar) in 52 effusions from dogs and cats.

BACKGROUND: Cell blocks are alternative preparations of fluid cytological specimens. They can be used for immunochemical studies as complementary tools or when other techniques (eg, immunocytochemistry, flow cytometry) are not available. OBJECTIVES: We aimed to provide comparative morphologic, immunohistochemical, and technical features of agar-based cell blocks (ACBs) and cell tube blocks (CTBs) from cavitary effusions. METHODS: Agar-based cell blocks and CTBs were obtained from canine and feline effusions with neoplastic/atypical cells or with packed cell volumes ≥3%. Cellularity, RBC separation, and cellular features were evaluated on digitalized H&E slides with evaluators blinded to the method. The immunohistochemical intensity and nonspecific background were assessed on pan-cytokeratin and vimentin-stained slides. Overall yield was calculated, and morphologic and immunohistochemical features were compared among paired samples. Technical and cellular features were also described. RESULTS: Agar-based cell blocks and CTBs yielded evaluable sections in 100% (52/52) and 98% (51/52) of the cases, respectively. Cellularity and RBC separation scores were significantly higher in CTBs. Similar staining intensities were observed, and background staining was more frequently seen in pan-cytokeratin-stained ACBs. Only basic materials and equipment were required for both methods. Agar-based cell block preparations were more operator dependent and difficult to standardize, whereas CTBs were easier to prepare, but laboratory processing was more demanding. CONCLUSIONS: Both methods can be used to produce good sections for immunohistochemistry staining with no significant differences. Cell tube blocks are beneficial for RBC-rich samples, and little additional training is required to prepare the blocks. Both types of cell blocks are reliable, cost-effective methods that could be introduced in diagnostic laboratories to further characterize canine and feline effusions.

It is all in the bag: collodion bag versus HistoGel cell block method.

INTRODUCTION: We performed a comparison of cell blocks prepared with the collodion bag and HistoGel to evaluate the ease of embedding and cutting, performance with low cellularity specimens, time and cost per specimen, and value to support immunohistochemistry and molecular diagnostics. MATERIALS AND METHODS: We processed 11 fresh, unfixed effusions using both the collodion bag and the HistoGel cell block preparation methods. Six immunohistochemistry stains were tested on 2 of the body fluids. DNA was extracted and quantified, and polymerase chain reaction cycle thresholds were evaluated from cell blocks prepared from 5 of the body fluids. The comparison parameters included embedding difficulty, cutting resistance, adequacy, cell yield, cell preservation, immunohistochemistry stain quality, DNA quantity, integrity, and purity. The time and cost to prepare each specimen was compared using normalized values for preparation of specimen, cost per year, and cost per specimen. RESULTS: Each parameter was assessed for both cell block preparation methods. All 3 of the samples with moderate or poor cell yield were low-volume (5-mL) samples prepared with the HistoGel method. In contrast, the collodion bag method produced a good yield with all three 5-mL samples. DNA recovery was greater in the collodion bag method. Similar crossing threshold values in purity reactions indicated equally high-quality matrix properties for the collodion bag and HistoGel preparations. Preparation of the specimen was 10 minutes faster with the collodion bag method, and the cost for the collodion bag method was $0.24 more expensive per cell block than using the HistoGel. CONCLUSIONS: The collodion bag method produced superior cell blocks for both morphologic and molecular studies more consistently, with lower volume specimens and with less time per specimen.

Comparison of plasma-thrombin, HistoGel, and CellGel cell block preparation methods with paired ThinPrep slides in the setting of mediastinal granulomatous disease.

INTRODUCTION: Various cell block (CB) preparation methods are utilized by different laboratories, and not all laboratories perform CBs in tandem with ThinPreps (TPs). To compare the performance of different CB methods and their diagnostic value when used in conjunction with TP, we assessed the quantity and size of granulomas obtained from endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) of lymph nodes in the evaluation of granulomatous mediastinal disease. MATERIALS AND METHODS: A retrospective analysis of mediastinal lymph node EBUS-TBNA specimens that detected granulomas at our institution was performed. A total of 264 specimens from 124 patients had a TP followed by a CB (either plasma-thrombin, HistoGel, or CellGel) prepared from the residual material in the PreservCyt vial. The number and size of granulomas on each preparation was assessed using digital software. RESULTS: Granulomas were detected only on the CB in 18.9% of cases and only on the TP in 5.3%. All 3 CB preparation methods showed significantly more and larger granulomas compared with the paired TP, with the plasma-thrombin and CellGel methods yielding more diagnostic material than the HistoGel method. In addition, the average number of granulomas (4.0 ± 0.4 versus 15.3 ± 1.1) and granuloma size (119.2 ± 3.2 µm versus 271.8 ± 7.3 µm) were significantly lower on TP compared with CB, respectively. CONCLUSIONS: Plasma-thrombin and CellGel CB preparation methods had a higher granuloma yield compared with the HistoGel method. Additionally, significantly more numerous and larger granulomas were present on CBs compared with TP slides. Therefore, solely relying on TP slide evaluation may unintentionally overlook larger tissue fragments obtained during needle aspirations.

Natural Histogel-Based Bio-Scaffolds for Sustaining Angiogenesis in Beige Adipose Tissue.

In the treatment of obesity and its related disorders, one of the measures adopted is weight reduction by controlling nutrition and increasing physical activity. A valid alternative to restore the physiological function of the human body could be the increase of energy consumption by inducing the browning of adipose tissue. To this purpose, we tested the ability of Histogel, a natural mixture of glycosaminoglycans isolated from animal Wharton jelly, to sustain the differentiation of adipose derived mesenchymal cells (ADSCs) into brown-like cells expressing UCP-1. Differentiated cells show a higher energy metabolism compared to undifferentiated mesenchymal cells. Furthermore, Histogel acts as a pro-angiogenic matrix, induces endothelial cell proliferation and sprouting in a three-dimensional gel in vitro, and stimulates neovascularization when applied in vivo on top of the chicken embryo chorioallantoic membrane or injected subcutaneously in mice. In addition to the pro-angiogenic activity of Histogel, also the ADSC derived beige cells contribute to activating endothelial cells. These data led us to propose Histogel as a promising scaffold for the modulation of the thermogenic behavior of adipose tissue. Indeed, Histogel simultaneously supports the acquisition of brown tissue markers and activates the vasculature process necessary for the correct function of the thermogenic tissue. Thus, Histogel represents a valid candidate for the development of bioscaffolds to increase the amount of brown adipose tissue in patients with metabolic disorders.

Novel disposable cell block processing device and method for high cellular yield.

BACKGROUND: Cell blocks represent one of several cytology preparations, and play a significant role in providing tissue for ancillary studies, including immunohistochemistry and molecular testing. To the authors' knowledge, there currently is no standardized cell block processing method, and cellular yield often is suboptimal. To overcome the shortcomings of available methods, the authors developed a novel disposable cell block device, XCellent, that uses centrifugation to collect cells into a sample chamber. The sample chamber contains and protects the specimen during tissue processing and also is embedded and sectioned. The primary objectives of the current study were to compare cellular yield, cytomorphological detail, and processing times of HistoGel and XCellent. METHODS: Split sample testing of primarily fluid specimens was performed. Group 1 comprised large-volume and/or cloudy fluids and group 2 was residual samples in PreservCyt solution. Two cell blocks, 1 HistoGel cell block and 1 XCellent cell block, were processed from each sample. Cellularity was assessed using computerized image analysis and cytomorphology from 1+ to 3+. Processing times for 16 cell blocks also were recorded. RESULTS: A total of 50 cell blocks (25 HistoGel samples and 25 XCellent samples) were prepared. On average, cell blocks prepared using XCellent were 371% more cellular than those prepared with HistoGel and had better cytomorphology. Less preparation time was required for bulk processing of XCellent cell blocks (11 minutes) compared with the same quantity of bulk processed using HistoGel (38 minutes). CONCLUSIONS: XCellent provides an alternative cell block processing technique that offers high cellular yield and cytomorphology. In addition, the method uses the standard centrifugation process, which integrates into the existing cytology laboratory workflow.

A superior method for cell block preparation for fine-needle aspiration biopsies.

BACKGROUND: Cell block (CB) techniques for fine-needle aspiration biopsies (FNABs) vary. A direct comparison of CB techniques with statistical validation was performed to identify the best method. METHODS: Three CB techniques were compared: 1) FNAB rinsed in saline and clotted with plasma and thrombin (SPT); 2) FNAB rinsed in formalin and clotted with HistoGel (HG); and 3) FNAB rinsed in formalin, centrifuged, and the pellet captured in a collodion bag (ColB). FNAB was performed on 35 random surgical specimens for smears and each CB technique. A randomized blinded review of hematoxylin and eosin-stained CB slides was performed and each case was scored on a scale of 1 to 3 for cellularity, preservation, and architecture and the overall best CB was identified. Significance was determined by the Mann-Whitney U test for nonparametric ordinal data. RESULTS: The mean cellularity score was 1.71 for SPT (standard deviation [SD], 0.89), 1.68 for HG (SD, 0.67), and 3.0 for ColB (SD, 0). The mean preservation score was 1.31 for SPT (SD, 0.58), 1.54 for HG (SD, 0.70), and 2.91 for ColB (SD, 0.37). The mean architecture score was 1.45 for SPT (SD, 0.70), 1.43 for HG (SD, 0.60), and 2.71 for ColB (SD, 0.57). There was no statistical significance noted between SPT or HG when compared for each category. ColB was found to be superior to both SPT and HG when compared for each category (P<.05). The overall best CB was obtained with ColB in 33 of 35 cases (94%), with SPT proving superior in 1 of 35 cases (3%) and HG superior in 1 of 35 cases (3%). CONCLUSIONS: ColB appears to be a superior technique for CB, yielding greater cellularity, preservation, and architecture in the majority of cases. Cancer Cytopathol 2016;124:508-18. © 2016 American Cancer Society.