Reference standards for gene fusion molecular assays on cytological samples: an international validation study

AimsGene fusions assays are key for personalised treatments of advanced human cancers. Their implementation on cytological material requires a preliminary validation that may make use of cell line slides mimicking cytological samples. In this international multi-institutional study, gene fusion reference standards were developed and validated.MethodsCell lines harbouring EML4(13)–ALK(20) and SLC34A2(4)–ROS1(32) gene fusions were adopted to prepare reference standards. Eight laboratories (five adopting amplicon-based and three hybridisation-based platforms) received, at different dilution points two sets of slides (slide A 50.0%, slide B 25.0%, slide C 12.5% and slide D wild type) stained by Papanicolaou (Pap) and May Grunwald Giemsa (MGG). Analysis was carried out on a total of 64 slides.ResultsFour (50.0%) out of eight laboratories reported results on all slides and dilution points. While 12 (37.5%) out of 32 MGG slides were inadequate, 27 (84.4%) out of 32 Pap slides produced libraries adequate for variant calling. The laboratories using hybridisation-based platforms showed the highest rate of inadequate results (13/24 slides, 54.2%). Conversely, only 10.0% (4/40 slides) of inadequate results were reported by laboratories adopting amplicon-based platforms.ConclusionsReference standards in cytological format yield better results when Pap staining and processed by amplicon-based assays. Further investigation is required to optimise these standards for MGG stained cells and for hybridisation-based approaches.

Diagnostic Efficacy of DNA Ploidy in Liquid Based Cervical Cytology using DNA Cytometry

Worldwide cervical cancer is the fourth most common cancer in women and high incidence is reported from India. Liquid Based Cytology (LBC) provides good morphology for detection of cellular abnormalities. We, therefore, reviewed diagnostic efficacy of conventional Pap staining, flow cytometry and Human Papilloma Virus (HPV) testing in cervical pre cancer and cancer. Narrative review of cervical pre cancer and cancer candidate biomarkers including Pap staining, HPV and flow cytometry from cervical cytology fluids, is based on a detailed review of the literature. Based on the so far conducted studies, a promising conclusion can be drawn, that cytometry when coupled with HPV DNA typing or the conventional cytology gives better results as compared to that of conventional cytology or DNA cytometry alone. Liquid cytology provides a good and stable source of cervical cells to carry out ploidy studies using DNA cytometry. The procedure should be used in conjunction with LBC and HPV detection.

Application of Subtype-Specific Monoclonal Antibodies for Rapid Detection and Identification of Influenza A and B Viruses

We established a rapid method for the identification of influenza A and B virus strains: the peroxidase-antiperoxidase (PAP) staining method with two subtype-specific murine monoclonal antibodies, C179 (H1 and H2 specific) and F49 (H3 specific), and an anti-influenza B virus rabbit polyclonal serum. The types and subtypes of 160 strains were examined, and 158 strains were identified to be the same by the hemagglutination-inhibition (HI) test and the PAP method. In contrast to the results by the HI test, two strains were revealed to be a mixture of two subtypes (H1 and H3) by the PAP method, which was confirmed by plaque cloning. We further analyzed clinical specimens by the PAP method by directly inoculating specimens into Madin-Darby canine kidney cells in microplates. After 40 h of incubation, the types and subtypes of viruses in 52 of 152 specimens were clearly identified. Since the reactivities of the two monoclonal antibodies are not influenced by the antigenic drift of influenza virus, the newly developed method should be applicable not only for rapid diagnosis but also for the epidemiological study of influenza.

Practical Methods for TEM, Part II.

This is an extension of our presentation from last year. We intend to continue the discussion and review several practical procedures developed in our laboratory for routine and immuno-electron microscopy.TEM Vs IM-LM In the last decade, the use of diagnostic TEM in the medical field has experienced a drastic decline. The main reason is due to the advances of immuno-labeling techniques for light microscopy and also the decreased coverage of medical insurance for EM. Currently, immuno-histochemistry or in situ hybridization staining can be performed on large paraffin or frozen sections. These procedures can be performed quickly and much less expensively than TEM. However, TEM is still needed when PAP staining is not specific or minimum numbers of cells are stained. It is also possible to process immuno-labeled paraffin slides for TEM examination to confirm a diagnosis. In addition, when only a few microorganisms such as pneumocystic carinii or protothecosis are stained by Grocott’s methenamine silver in paraffin sections, a re-examination and confirmation by TEM can be performed without the use of uranium or lead staining.

PSA-negative tumours of the prostate

The immunoperoxidase staining of normal and hyperplastic prostatic epithelium for Prostate Specific Antigen (PSA) is, with few exceptions, uniform and strong. In contrast to the benign tissue, most reports of prostatic adenocarcinoma demonstrated an apparent correlation between staining-variability and increasing tumour grades. Decreased PAP staining may be related to incorrect manipulation of prostatic tissue. Finally there are many tumours and pseudotumours of the prostate in which the PSA stain is regularly negative. In the differential diagnosis of prostate tumour, both PSA and PAP (Prostatic Acid Phosphatase) stains should be performed. It is mandatory that the stains be interpreted in the appropriate histologic and clinicopathologic setting with due consideration of positive and negative staining.

Multimarker immunohistochemical staining of calgranulins, chloroacetate esterase, and S100 for simultaneous demonstration of inflammatory cells on paraffin sections.

Mac387 monoclonal antibody (MAb) recognizes two calcium binding, myeloid-associated proteins, now termed calgranulins, expressed at high levels by neutrophils and monocytes. Calgranulins are related to migration inhibitory factor (MIF) and are lost in a few days from monocytes differentiated in vitro. This marker is therefore potentially useful to analyze macrophage heterogeneity and turnover in tissue sections. In this study, we developed an immunohistochemical multimarker technique, including calgranulin demonstration, suitable for analyzing different inflammatory cells on paraffin-embedded material. The technique was carried out in subsequent steps demonstrating (a) naphthol AS-D chloroacetate esterase (CAE); (b) S100 immunoreactivity using a rabbit antibody in peroxidase-antiperoxidase (PAP) staining; and (c) Mac387 immunoreactivity using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique. CAE staining was introduced in this method to distinguish Mac387+/CAE- macrophages from Mac387+/CAE+ neutrophils, and Mac387-/CAE+ mast cells. S100 protein is strongly expressed within lymphoid tissues by dendritic accessory cells and was then applied to distinguish these cells from S100-macrophages. We have also verified the possibility of reducing the staining time for this time-consuming procedure by use of microwave irradiation. The technique was applied to a representative variety of normal and pathological samples to assess its usefulness for study of cell heterogeneity. Our results showed the multimarker technique to be highly informative in the study of inflammatory lesions (e.g., rheumatoid arthritis, sarcoid and cat-scratch granulomas, dermathopathic lymphadenopathy), and is of wide potential value as an aid to histopathological diagnosis of several diseases.

Immunolocalization of Type VI collagen in calfskin: A correlative study

Although fine details of the fundamental structural units of calfskin, the types I and III collagen fibrils, have been well elucidated, little is known about the location of “minority” collagens in the skin before and after processing to leather. Using microscopic methods, we investigated one such collagen, type VI, in calfskin to determine its presence and distribution.Four different methods of identification of type VI collagen were employed: immunofluorescence and peroxidase-antiperoxidase (PAP) staining using light microscopy, and transmission (TEM) and scanning (SEM) immunoelectron microscopy. In all cases, the primary antibody used was a polyclonal anti-type VI collagen raised in rabbitss. For light microscopy, 12 μm frozen sections were prepared. Fluorescent labeling was carried out with anti-rabbit IgG conjugated to r-phycoerythrin as secondary antibody. PAP staining was performed with the modified techniques of Amenta et al. Tissue for SEM was prepared by a modified protocol of Keene et al. without prefixation: the secondary antibody used was goat anti-rabbit IgG conjugated to 30 nm colloidal gold; it was then fixed in glutaraldehyde-paraformaldehyde and OsO4, dehydrated, critical point dried and sputter coated with gold.

Detection of intracytoplasmic immunoglobulin by flow cytometry in B-cell malignancies.

The aim of this study was to compare the results of flow cytometric (FCM) determination of heavy and light chain cytoplasmic immunoglobulin (cIg) with those obtained by the peroxidase-antiperoxidase (PAP) method. Fifty-one patients, including five non-T-acute lymphoblastic leukemias, 16 B-chronic lymphocytic leukemias (CLL), 13 non-Hodgkin's lymphomas, seven hairy cell leukemias, four multiple myeloma/plasma cell leukemias, and six T-cell leukemia/lymphomas, as well as 12 normal controls, were studied. Saponin-permeabilized cell suspensions were indirectly stained with monoclonal antibodies and analyzed by flow cytometry. Acetone-fixed cytocentrifuge smears were stained for cIg by the PAP method. The results obtained indicate that: (a) detection of cIg by FCM is a feasible and useful technique to confirm the B-cell lineage of leukemias and lymphomas, particularly those characterized by low-density surface immunoglobulin, such as CLL; and (b) cIg detection by FCM and PAP staining are complementary methods to recognize with certainty the monoclonality of B-cell malignancies.