The value of immunohistochemistry in increasing diagnostic precision of undifferentiated tumours by the surgical pathologist

Summary The availability of polyclonal and monoclonal antibodies allows immunohistochemical staining (immunofluorescence and immunoperoxidase) procedures to be used by the surgical pathologist, so as to obtain a definite diagnosis in cases where light microscopic examination of tissue sections from biopsy and surgical specimens is inconclusive. Four cases of undifferentiated tumors are described in which only a differential diagnosis could be obtained using light microscopy but a definite diagnosis was achieved when additional information was provided by immunohistochemistry. A scheme is presented for the use of immunohistochemistry to increase diagnostic precision in assessing tumours.     

Classification of routinely processed anaplastic large cell tumours with a small panel of antibodies. An immunohistochemical study with clinical follow-up

A proportion of anaplastic large cell tumours is difficult to classify on sections of routinely processed, paraffin-embedded tissue. Differentiation into large cell lymphoma, carcinoma, melanoma or sarcoma is important in order to assess prognosis and proper treatment. Although the use of immunohistochemistry has been reported in the differentiation between some of these types of neoplasms, no antibody panel, which can directly differentiate all of them, has been described. In the present study we evaluated the value of a panel of 5 antibodies for the classification of 29 anaplastic large cell tumours, which could not be classified by experienced pathologists using conventional histological and histochemical techniques. The panel, which can be used on routinely fixed paraffin-embedded tissue, consisted of 5 different antibodies directed against keratin, vimentin, the human milk-fat globule membrane antigen MAM-6, a melanoma associated antigen and common leucocyte antigen. The use of this panel directly resulted in a definite diagnosis in 95% of the cases and provided valuable information for the diagnosis in the remaining cases. The diagnosis was confirmed by additional marker studies and electron microscopy. Moreover, clinical follow-up, including treatment data, was in accordance with the diagnosis based on the panel.     

Non-surgical biopsy in lung cancer: a paradigm shift

Histological subgroups of non-small cell lung cancer have different prognosis and they require different therapeutic approaches. Accordingly, there is a clinical need in this field to supplement conventional pathological diagnostics with protein and genetic biomarkers that can help to recognize patients responsive to these therapies. Methods for subgroup classification and target identification were developed using surgical samples (surgical lung tumor specimens are available only in 20% of all lung cancer cases). The majority of lung cancer patients, however, have tumors that are irresectable at the time of diagnosis. Therefore, their diagnosis is usually based on bronchoscopically removed tissue or needle biopsy samples analyzed mainly by cytology. Because of the growing need for immunohistochemistry and molecular pathology in lung cancer diagnosis, emphasis should be given to diagnostic bronchoscopic procedures providing tissue samples. Combination of the different biopsy techniques (histology, cytology, bronchial brush, BAL, TBNA etc.), embedding the cells (preparing cell blocks) and, moreover, the availability of immunohistochemical and molecular pathological facilities are all required to set up the proper diagnosis and therapeutic strategy in human lung cancer. Strausz J, Tímár J. Non-surgical biopsy in lung cancer: a paradigm shift.     

Cytology and immunocytochemistry of bronchioloalveolar carcinoma

A study of the value of cytologic examination in the diagnosis of bronchioloalveolar carcinoma showed that fine needle aspiration (FNA) cytology is a definitive means of making the diagnosis of bronchioloalveolar carcinoma. In experienced hands, FNA cytology approached a diagnostic accuracy of 100%. With exfoliative cytology, technically adequate sputum samples offered an 82% diagnostic yield; sputum cytology should thus be considered as an adjunct method in establishing the diagnosis. Several cytologic features that strongly suggest the diagnosis of bronchioloalveolar carcinoma and help to differentiate it from adenocarcinomas of other body sites are outlined. It is also concluded that immunocytochemical stains have limited value in differentiating bronchioloalveolar carcinoma and other types of clear cell malignant neoplasms, including mesothelioma, unless a panel of antibodies is used, and are of no value in ascertaining whether the cells are benign or malignant.     

Schwannoma of ascending colon treated by laparoscopic right hemicolectomy

ABSTRACT: Schwannomas of the colon are rare and are difficult to diagnose preoperatively, since they often defy endoscopic and radiographic detection. Immunohistochemical stains are useful postoperatively to confirm this tumor, but more reliable diagnostic techniques (such as colonoscopic biopsy with immunohistochemistry) have emerged to enhance preoperative diagnostic accuracy. Here we report an instance of schwannoma arising in the ascending colon, where immunohistochemical staining of a preoperative biopsy facilitated diagnosis. After laparoscopic resection, histologic examination was confirmatory.     

Immunohistochemical antigen demonstration in plastic-embedded lymphoid tissue

We describe a method for post-embedding immunohistochemical demonstration of a wide range of antigens in glycol methacrylate-embedded tissue. Rat spleen and thymus tissues were fixed by immersion in fixatives containing different concentrations of paraformaldehyde, washed in sucrose phosphate buffer, dehydrated in acetone, infiltrated in a glycol methacrylate mixture in which the commonly used softener 2-butoxyethanol was replaced by butaandiol monoacrylate, and embedded. Trypsin was used to re-expose the masked antigenicity. Excellent results were obtained with a panel of monoclonal antibodies (MoAbs) directed against T-cells, B-cells, Ia-positive cells, macrophages, follicular dendritic cells, and leucocyte common antigen-bearing cells. The method described combines exact localization of antigens with optimal tissue morphology.     

Is there a role for antibody testing in the diagnosis of invasive candidiasis?

During the last decades, the use of antibody tests for the diagnosis of invasive mycoses has declined as a consequence of the general belief that they are insensitive and non-specific. However, there is a clear evidence that antibodies can be detected in highly immunodeficient patients (such as bone marrow transplant recipients), and that those antibodies are useful for the diagnosis. Antibody tests are currently in use as diagnostic tools for some primary mycoses, such as the endemic mycoses, aspergilloma, allergic bronchopulmonary aspergilosis and sporothrichosis. For invasive candidiasis, diagnostic methods must differentiate Candida colonization of mucous membranes or superficial infection from tissue invasion by this microorganism. Substantial progress has been made in diagnosis of invasive candidiasis with the development of a variety of methods for the detection of antibodies and antigens. However, no single test has found widespread clinical use and there is a consensus that diagnosis based on a single specimen lacks sensitivity. It is necessary to test sequential samples taken while the patient is at greatest risk for developing invasive candidiasis to optimize the diagnosis. Results obtained from a panel of diagnostic tests in association with clinical aspects will likely be the most useful strategy for early diagnosis and therapy.     

Evaluation of anti-human antibodies for immunohistochemistry on archival nonhuman primate tissues

Abstract: A panel of commercially available antibodies which recognize specific antigens on human tissues was developed for use in immunohistochemistry on tissues from eight species of nonhuman primates. Antibodies were selected for potential usefulness in diagnostic pathology, and for effectiveness in formalin-fixed, paraffin-embedded tissues. Tissues from four species of macaques and four New World monkeys were evaluated. Using these antibodies we were able to identify 17/21 antigens examined in all eight species, and 21/21 antigens in the four species of macaques. Detailed immunohistochemistry protocols are presented, along with a systematic approach to developing a protocol for a new antibody.     

Immunohistochemical Typing of Adenocarcinomas of the Pancreatobiliary System Improves Diagnosis and Prognostic Stratification

MethodsThis was a retrospective observational cohort study on patients with adenocarcinoma of the pancreatobiliary system who underwent diagnostic core needle biopsy or surgical resection at a tertiary referral center. 409 tumor samples were analyzed with up to 27 conventional antibodies used in diagnostic pathology. Immunohistochemical scoring system was the percentage of stained tumor cells. Bioinformatic analysis, internal validation, and survival analysis were performed.ResultsHierarchical clustering and differential expression analysis identified three immunohistochemical tumor types (extrahepatic pancreatobiliary, intestinal, and intrahepatic cholangiocarcinoma) and the discriminant markers between them. Among patients who underwent surgical resection of their primary tumor with curative intent, the intestinal type showed an adjusted hazard ratio of 0.19 for overall survival (95% confidence interval 0.05–0.72; p value = 0.014) compared to the extrahepatic pancreatobiliary type.ConclusionsIntegrative immunohistochemical classification of adenocarcinomas of the pancreatobiliary system results in a characteristic immunohistochemical profile for intrahepatic cholangiocarcinoma and intestinal type adenocarcinoma, which helps in distinguishing them from metastatic and pancreatobiliary type adenocarcinoma, respectively. A diagnostic immunohistochemical panel and additional extended panels of discriminant markers are proposed as guidance for their pathological diagnosis.     

Fluorescent immunohistochemistry and in situ hybridization analysis of mouse pancreas using low-power antigen-retrieval technique.

To facilitate the immunological reaction of antibodies with antigens in fixed tissues, it is necessary to unmask or retrieve the antigens through pretreatment of the specimens. However, adjustment of heating-induced antigen retrieval is always required for different tissues and antigens. In this study, by using a low-power antigen-retrieval technique with appropriate dilution of antibodies, we successfully immunostained key antigens in pancreas such as insulin, PDX-1, glucagon, cytokeratin, and CD31, which have previously presented a particular challenge for investigators because of the rapid autodigestion and high nonspecific antibody binding in this tissue. Satisfactory results were obtained when immunohistochemistry and fluorescence in situ hybridization analysis were combined in the same slides.     

Immunofluorescent Staining of Epon Embedded Kidney Sections Through Simultaneous use of Two Different Fluorochrome-Conjugated Antisera

Kidney biopsies can be examined in Epon sections for comparison of immunofluorescence and histology. This is possible by an incubation method which has now been modified to allow simultaneous localization of two antigens using fluorescein and rhodamine-conjugated antibodies on the same semithin sections of formalin fixed tissue. Consecutive sections from the same blocks can also be cut for electron microscopy. The method is now used in our immunopathological diagnostic procedures for examination of kidney biopsies.     

Effects of Prolonged Formalin Fixation on Diagnostic Immunohistochemistry in Domestic Animals

Immunohistochemistry (IHC) is routinely used in diagnostic pathology to detect infectious agents, to immunophenotype neoplastic cells, and to prognosticate neoplastic diseases. Formalin fixation is considered a limiting factor for IHC because formalin can cross-link antigens and mask epitopes. Prolonged formalin fixation is presumed to result in decreased antigen detection; however, this effect has only been evaluated with a few antibodies. The goal of this study was to evaluate the effect of prolonged formalin fixation on the immunohistochemical detection of 61 different antigens. Approximately 5-mm-thick tissue slices were fixed in 10% neutral-buffered formalin. Tissue slices were removed from formalin, processed, and paraffin-embedded at 1-day, 3-day, and then at ∼1-week intervals. IHC was performed on all sections in tandem after all tissues were processed. Immunoreactions were evaluated by three pathologists according to a four-tier grading system. Immunoreactivity of cytokeratin 7, high-molecular-weight cytokeratin, and laminin was diminished by prolonged formalin fixation. However, immunohistochemical reactivity remained moderate to strong with up to 7 weeks of fixation for all other antibodies. These results suggest that prolonged formalin fixation has minimal effects on antigen detection for most commonly used antibodies. These results further validate the use of IHC in diagnostic pathology. (J Histochem Cytochem 57:753–761, 2009)     

A novel technology allowing immunohistochemical staining of a tissue section with 50 different antibodies in a single experiment.

Immunohistochemical (IHC) examination is frequently necessary for a histological differential diagnosis of tumors. To simplify IHC examination, we have developed a novel device called a "multiplex-immunostain chip (MI chip)." The chip is a panel of antibodies contained in a silicon rubber plate that consists of 50 2-mm-diameter wells. A tissue section slide is placed on the plate and is fastened tightly with a specially designed clamp. The plate with the slide is then turned upside down, which applies the antibodies to the section. This technology allows IHC staining of a tissue section with 50 different antibodies in a single experiment, reducing the time, effort, and expense of IHC analysis. In addition, it enables pathologists to compare expression of multiple antigens on a tissue section simply by changing microscopic fields on a single slide. These features are unique to the MI chip technology. The method requires no expensive instruments. This device can be used in various applications in differential diagnosis of tumors and the field of cell biology.     

Immunofluorescent staining of Epon embedded kidney sections through simultaneous use of two different fluorochrome-conjugated antisera

Kidney biopsies can be examined in Epon sections for comparison of immunofluorescence and histology. This is possible by an incubation method which has now been modified to allow simultaneous localization of two antigens using fluorescein and rhodamine-conjugated antibodies on the same semithin sections of formalin fixed tissue. Consecutive sections from the same blocks can also be cut for electron microscopy. The method is now used in our immunopathological diagnostic procedures for examination of kidney biopsies.     

Immunohistochemical profiling of the ultimobranchial remnants in the rat postnatal thyroid gland

Ultimobranchial (UB) remnants are a constant presence in the thyroid throughout rat postnatal life; however, the difficulty in identifying the most immature forms from the surrounding thyroid tissue prompted us to search for a specific marker. With that objective, we applied a panel of antibodies reported to be specific for their human counterpart, solid cell nests (SCNs), using double immunohistochemistry and immunofluorescence. Our results demonstrated that cytokeratin 34βE12 and p63 are highly sensitive markers for the immunohistologic screening of UB‐remnants, independently of their maturity or size. Furthermore, rat UB‐follicles (UBFs) coincided with human SCNs in the immunohistochemical pattern exhibited by both antigens. In contrast, the pattern displayed for calcitonin and thyroglobulin differs considerably but confirm the hypothesis that rat UB‐cells can differentiate into both types of thyroid endocrine cells. This hypothesis agrees with recent findings that thyroid C‐cells share an endodermic origin with follicular cells in rodents. We suggest that the persistence of p63‐positive undifferentiated cells in UB‐remnants may constitute a reservoir of basal/stem cells that persist beyond embryogenesis from which, in certain unknown conditions, differentiated thyroid cells or even unusual tumors may arise.     

Histology in diagnosis of parasitic diseases

Many pathogenic organisms cause inflammatory lesions and microscopic findings are a useful diagnostic tool for the aetiological diagnosis. However, the histological lesions are limited in respect to many biological agents that can damage the tissues. The histologic hallmark of parasitic diseases is mostly granulomatous inflammation. It is characterized by a focal infiltration of macrophages and epithelioid cells. Many giant cells, lymphocytes, plasma cells, fibroblasts and granulocytes can be found. Agents inducing granulomas include helminths and parasites that replicate intracellularly. Some special stains are utilized in histopathology, for example Giemsa's stain is useful to identify Leishmania. Using specific antibodies, immunohistochemical methods provide an aetiological diagnosis. Sometimes, tissue damage can be immuno-mediated depending on deposit of circulating immunocomplexes or T-lymphocytes involvement rather than by direct parasitic injury. Generally, the lesions which can be observed are respectively vasculitis and inflammatory reactions predominantly composed of mononuclear cells, as observed in many viral or bacterial diseases. In these cases, aetiological diagnosis is improved by in situ-PCR. For microscopic identification of parasites in tissues it is also important to be familiar with the kind of parasites most likely to be found in the examined tissue and in that particular host. Localization of parasites can induce hyperplastic-neoplastic lesions. Many parasites have been associated with the occurrence of specific types of neoplasms, but the mechanisms involved are still not well defined. Chronic inflammation and/or immune suppression seem to induce neoplastic proliferation.     

流行性出血热患者皮肤组织内病毒抗原及免疫复合物检测与意义

应用常规病理、免疫病理及超微病理技术,对３３例流行性出血热（ＥＨＦ）患者皮肤活检标本的病理变化及病毒抗原、免疫复合物进行观察,同时与血清病毒抗原、抗体及循环免疫复合物检出情况进行比较。在２３例ＥＨＦ患者皮肤微血管内皮细胞中检出病毒抗原,部分组织中可同时检出免疫球蛋白及Ｃ３,少数组织仅能检出病毒抗原或免疫球蛋白。配对血清小也可检出ＥＨＦ病毒抗原、抗体及循环免疫复合物。组织及血清免疫复合物形成与血清补体Ｃ３水平下降有关,组织内肥大细胞脱颗粒与血清ＩｇＥ水平升高相关,提示多种变态反应参与了流行性出血热的发病机制。     

Reclassification of gastrointestinal mesenchymal tumors

A retrospective (1985-2005) multicentric evaluation of the pathological diagnoses of GIST (gastrointestinal stromal tumor) in Hungary was performed. A large cohort of 463 patients with abdominal mesenchymal tumors was reevaluated with the help of immunohistochemistry for CD117, CD34, desmin and S100. Prognostic groups have been established based on international criteria. Two hundred fifty five GISTs have been found in 245 patients during the evaluated period. Beside GIST, 81 leiomyogenic and 25 neurogenic tumors were the primary pathological diagnosis and in 74/255 cases the primary diagnosis was a benign tumor. In our cohort high-risk GIST cases were found to be the predominant. Based on our experience, in case of abdominal tumors of uncertain localization, immunohistochemical markers of CD117, CD34, S100 and desmin provide a proper tool for precise identification of the tumor entity including GIST.     

Mastocytosis in children: clinicopathological study based on 35 cases

Immunohistochemical staining is useful in the diagnosis of bone marrow infiltration in systemic mastocytosis. However, it is not clear if antibody staining may be helpful in the diagnosis of cutaneous mastocytosis (CM). We studied the histological appearance of CM in 35 pediatric patients. Cases were assigned to three basic clinical groups: I--Urticaria pigmentosa (UP, n=29); II--Mastocytomas (n=4); and III--Diffuse Cutaneous Mastocytosis (DCM, n=2). The analysis of clinical information revealed an association between the presence of diarrhea and a higher number of cells/field. Nine doubtful cases, all of them macules, were selected based on the scarcity of mast cells (MC) and the absence or rarity of other inflammatory cells. We compared the number of cells identified in Giemsa and immunohistochemical stains in definite and doubtful cases. The intraclass correlation statistic tested the concordance between each staining method. All 9 dubious cases according to the Giemsa stain had their CM diagnosis confirmed by the immunohistochemistry analysis. The intraclass correlation between Giemsa and c-kit was good (0.7) when the number of MC was high. However, there was no correlation between the mast cells counts in the two different stains in the dubious cases. The immunohistochemistry with c-kit might make CM diagnosis easier, especially in the macular cases, when there is a lower number of MC.