Immunofluorescence analysis of normal and malignant lymphoid tissues with selected combination of antisera

Tissue sections stained with combinations of antisera labeled with different fluorochromes (i.e., conventional antisera to human immunoglobulin classes, T lymphocyte antigens, and Ia-like p28,33 antigens used in various double combinations with each other or with different mouse monoclonal antibodies) allow the identification of the different areas of lymph nodes in serial sections and provide great flexibility as well as precision in the analysis of the distribution and relationship of normal and malignant cells. Lymphoid microenvironments in the thymus and the paracortical areas of lymph nodes are described. The close association of T lymphocytes and nonlymphoid cells expressing large amounts of Ia-like antigens (such as interdigitating reticular cells and endothelium) may be relevant for the understanding of immunoregulatory disorders such as dermatopathic and angioimmunoblastic lymphadenopathies and some malignancies (e.g., mycosis fungoides) were the expression of Ia-like antigens on non-T cells seems to be abnormally abundant. The analysis of immunoglobulin and membrane marker expression of normal and malignant B cells and their relation to T cells can also be related to the histology of the disease. These studies are clinically useful for the classification of childhood lymphomas, the differential diagnosis of anaplastic carcinomas and lymphomas, and in the study of the early stages of lymphomas.     

Cytomorphology and immunocytochemistry of fine needle aspirates from blastic non-Hodgkin's lymphomas

Fine needle aspirates from 54 consecutive patients with primary or recurrent blastic (high-grade malignant) non-Hodgkin's lymphomas (NHLs) were analyzed by cytomorphology and immunocytochemistry. The cytologic diagnoses induced follicular center-cell-derived (centroblastic or anaplastic centrocytic) lymphoma (31 cases), immunoblastic lymphoma (11 cases), lymphoblastic lymphoma (9 cases) and histiocytic lymphoma (3 cases). Immunocytochemistry showed a B-cell phenotype of the neoplastic lymphocytes in all lymphoblastic lymphomas, 29 follicle center-cell lymphomas and 4 immunoblastic lymphomas. Four of the immunoblastic lymphomas were of T-cell origin while one case was not evaluable due to necrosis. A histiocytic origin was confirmed in two of the three cases that had a cytologic diagnosis of histiocytic lymphoma; the third case was shown by immunocytochemistry to be a true Ki-1-positive large cell lymphoma. Histologic and immunohistochemical analysis were performed on surgical biopsies from 18 patients. The results were in agreement with those on the fine needle aspiration (FNA) material in 14 cases. Three lymphomas could be phenotyped on aspirated material while marker studies on excised material were inconclusive. One lymph node aspirate contained mostly necrotic cells, which were unsatisfactory for adequate immunocytochemistry. However, sections from a removed tonsil from the same patient could be used for conclusive histology and phenotyping. In conclusion, the high diagnostic accuracy of combined cytomorphologic and immunocytochemical assessment of FNA samples validates the use of the technique in the diagnostic work-up of blastic (high-grade malignant) NHLs. In fact, the diagnostic accuracy seems so high that the technique can safely be used in the final diagnosis of blastic NHLs.     

A comparison of fixation methods on lung morphology in a murine model of emphysema

Emphysema is characterized by enlargement of the alveoli, which is the most important parameter to assess the presence and severity of this disease. Alveolar enlargement is primarily defined on morphological criteria; therefore, characterization of this disease with morphological parameters is a prerequisite to study the pathogenesis. For this purpose, different methods of lung fixation were evaluated in a murine model of LPS-induced lung emphysema. Five different methods of lung fixation were evaluated: intratracheal instillation of fixatives, in situ fixation, fixed-volume fixation, vascular whole body perfusion, and vacuum inflation. In addition, the effects of three different fixatives (10% formalin, Carnoy's, and agarose/10% formalin solution) and two embedding methods (paraffin and plastic) were investigated on the murine lung morphology. Mice received intranasal administration of LPS to induce alveolar wall destruction. Quantification of air space enlargement was determined by mean linear intercept analysis, and the histological sections were analyzed for the most optimal fixation method. Additionally, routine immunohistological staining was performed on lung tissue of PBS-treated mice. Intratracheal instillation of formalin or agarose/formalin solution, in situ fixation, and fixed-volume fixation provided a normal lung architecture, in contrast to the lungs fixed via whole body perfusion and vacuum inflation. Formalin-fixed lungs resulted in the most optimal lung morphology for lung emphysema analysis when embedded in paraffin, while for Carnoy's fixed lungs, plastic embedding was preferred. The histological findings, the mean linear intercept measurement, and the immunohistochemistry data demonstrated that fixation by intratracheal instillation of 10% formalin or in situ fixation with 10% formalin are the two most optimal methods to fix lungs for alveolar enlargement analysis to study lung emphysema.     

Morphology and index of nucleoli in malignant lymphomas

The significance of morphology and the number of lymphocyte nucleoli was evaluated by electronmicroscopic sections of malignant lymphomas. It was not only the number and the size of nucleoli which characterized malignant lymphomas differing in histological respect, but above all the different morphology of the nucleoli. The prognostic significance of the nucleolus can be illustrated, if malignant lymphomas of a low grade malignancy with a preponderance of micronucleoli and a low nucleolar index are compared with a significantly higher nucleolar index and markedly increased macronucleoli. These differences are further supplemented by additional morphological properties of nucleoli in malignant lymphomas.     

The Status of Immunohistochemical Studies in Lymphoma Diagnosis

Immunohistochemical studies of formalin fixed paraffin embedded sections today constitute an essential component of the diagnostic process for malignant lymphoma. Lineage related markers are of value in distinguishing various lymphomas from anaplastic large and small cell carcinomas, and from sarcomas. Other more recently available markers are of value in establishing prognosis and, on occasion, in differentiating between neoplastic and reactive proliferations. Meticulous attention to reagent control and technical procedures is essential to all such studies.     

The Status of Immunohistochemical Studies in Lymphoma Diagnosis

Immunohistochemical studies of formalin fixed paraffin embedded sections today constitute an essential component of the diagnostic process for malignant lymphoma. Lineage related markers are of value in distinguishing various lymphomas from anaplastic large and small cell carcinomas, and from sarcomas. Other more recently available markers are of value in establishing prognosis and, on occasion, in differentiating between neoplastic and reactive proliferations. Meticulous attention to reagent control and technical procedures is essential to all such studies.     

Alteration of immunoglobulin-bearing lymphoma cells by fixation.

Four large cell lymphomas known to be monoclonal B-cell proliferations were studied with immunofluorescent and immunohistochemical methods for the detection of kappa- and lambda-light chains. Frozen sections of lymphoma tissues as well as formalin and B-5-fixed tissues embedded in paraffin were studied. Both immunofluorescent and immunohistochemical methods gave similar results on frozen sections; however, a number of discrepancies were noted between the results obtained on fixed tissues and those obtained on frozen tissues. In an effort to identify a fixative which did not alter immunoglobulin (Ig), mouse lymph nodes were fixed in different fixatives before Ig detection; but all of the fixatives tested destroyed the Ig present on normal cortical B lymphocytes. Immunoglobulin-bearing normal and neoplastic lymphocytes are better detected on frozen sections than on paraffin sections after routine fixation.     

Effects of prolonged water washing of tissue samples fixed in formalin on histological staining

The effects of prolonged water washing after fixation for 48 h in 10% (v/v) phosphate-buffered neutral formalin on the quality of representative histological staining methods were evaluated using samples of liver, kidney, spleen and thymus collected from three male Crl:CD(SD)(IGS) rats and one male beagle dog. Because door-to-door courier services in Japan prohibit handling formalin, our goal was to confirm that formalin fixed wet tissue samples could be stored in tap water rather than formalin during transportation of the samples without decreasing the quality of their staining or immunohistochemistry. Each tissue sample was allocated randomly to one of three groups: 12 min, 3 days and 7 days of washing in running tap water; samples then were routinely embedded in paraffin and sectioned. The sections were stained with hematoxylin and eosin, periodic acid-Schiff, azan, and the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method. Immunohistochemical staining for Factor VIII, ED-1 and CD3 also was assessed. Prolonged water washing for up to 7 days did not affect the morphology or stainability by standard histological methods, or the intensity and frequency of positive reactions using the TUNEL method. Only immunohistochemical staining of Factor VIII was altered in both the rat and dog sections after 7 days of water washing. The intensity of positive reactions of Factor VIII immunohistochemistry after 7 days water washing was still strong enough to detect microscopically. Therefore, prolonged water washing for up to 7 days after formalin fixation does not have seriously detrimental effects on the quality and characteristics of paraffin sections stained by various methods, including immunohistochemistry.     

Cytodiagnosis of malignant non-Hodgkin's lymphomas in effusions (author's transl)

Body cavity effusions in malignant non-Hodgkin's lymphomas, particularly in the early stages of those neoplasms, are rare in comparison to the far more common effusions in other malignant diseases and in inflammatory processes. Therefore, the cytological differential diagnosis is of great importance. Of 7,000 pleural and 1,700 ascitic effusions, only 42 cases were malignant non-Hodgkin's lymphoma and in 30 lymphoma was suspected. When lymphoma is suspected, particularly low-grade lymphoma, there are great difficulties in making a differential diagnosis. Using the more or less typical cellular and nuclear criteria of the various lymphoma types, an attempt was made to classify the unequivocal lymphomas according to the Kiel classification of malignant non-Hodgkin's lymphomas. In principle these criteria are the same in cytological and histological examinations. In 31 cases the lymphoma subtype could be specified and confirmed in part by subsequent histological examination. Apart from the suspect lymphoma cases (40%), cases of a low grade of malignancy were predominantly observed (28%). Lymphomas with a high grade of malignancy were less frequently encountered (15%), a proportion similar to that of unclassifiable lymphomas (16%). Apparently the cytological and karyological criteria are not yet adequate to classify lymphomas from conventionally prepared cytological specimens with a higher degree of accuracy.     

Cytogenetics in malignant lymphoma.

There appear to be four primary areas of interest in the application of cytogenetic techniques to the study of malignant lymphomas: (1) the role of cytogenetics in the diagnosis of lymphoma in problem cases, (2) as an aid to the classification of malignant lymphomas, (3) whether specific chromosomal patterns will have prognostic significance for response to therapy or survival, and (4) the role of cytogenetics in staging of malignant lymphomas. A case of reactive lymphoid hyperplasia is reported in which cytogenetic studies demonstrated an aneuploid clone suggesting that cytogenetic abnormalities of lymphoma may precede the diagnostic histopathologic picture. The occurrence of 14q+ marker chromosomes in plasmacytic myeloma, plasma cell leukemia, malignant lymphomas, Burkitt's lymphoma, and ataxia-telangiectasia suggest that a common etiologic or pathogenetic mechanism may be present in some of these disorders. A preliminary pilot study of spleens removed at staging laparotomy for Hdgkin's disease suggests that cytogenetic studies may be able to detect Hodgkin's disease that is not apparent histologically. Further studies are required to provide answers to these areas of interest in cytogenetics in malignant lymphoma.     

Cytochemical examination of acid phosphatase and beta-glucuronidase enzymes in low-grade B cell non-Hodgkin's lymphomas

The differential diagnostic significance of acid phosphatase and beta-glucuronidase were studied in 77 cases of low-grade B cell non-Hodgkin's lymphomas. In most cases the results of cytochemical enzyme studies performed on malignant cells of the bone marrow were evaluated. B cell chronic lymphocytic leukaemia, centrocytic and centroblastic/centrocytic lymphomas were characterized by a weak or a negative acid phosphatase and beta-glucuronidase activity. Stronger positivity was observed in immunocytoma and in Waldenstr?m's macroglobulinaemia, while the highest activity was found in multiple myeloma. Hairy cell leukaemia of B cell origin showed intensive tartrate-resistant acid phosphatase activity. The cytochemical examination of these lysosomal enzymes may be useful in the diagnosis of low-grade malignant lymphomas of B cell origin by completing other methods.     

Evaluation of the suitability of ex vivo handled ovarian tissues for optical diagnosis by Raman microspectroscopy

A pilot Raman microspectroscopy study of formalin-fixed, paraffin-embedded, and deparaffinized sections from the same ovarian normal and malignant tissues was carried out. This approach was considered in order to evaluate the suitability of these ex vivo tissue handling procedures in discrimination as well as biochemical characterization. The spectra of formalin-fixed normal and malignant tissues exhibited no contamination due to formalin, which is indicated by the absence of strong formalin peaks; spectral features also show significant differences for normal and malignant tissues. The differences between spectral profiles of deparaffinized normal and malignant tissues are subtle and spectra show few residual sharp peaks of paraffin. Complete dominance of paraffin swamping signals from tissues was observed in the spectra of paraffin-embedded tissues. Principal components analysis (PCA), which was employed for discrimination of tissue type, provided good discrimination for formalin-fixed and paraffin-embedded tissue spectra. PCA of deparaffinized tissues resulted in a poor classification with significant overlap among the clusters. Thus, this study indicates that formalin fixation is the most suitable among the three procedures employed in the study. Significant differences between spectral profiles of normal and malignant formalin-fixed tissues can not only be exploited for discrimination but can also provide information on biochemical characteristics of the tissues. Deparaffinized tissues provide poor discrimination and information on tissue biochemistry is lost. Paraffin-embedded tissues may provide good discrimination, but predominance of paraffin in the spectra could jeopardize biochemical characterization. Prospectively, as a result of the better availability of paraffin-embedded tissues and problems associated with frozen sectioning of formalin-fixed tissues, the results of this study using paraffin-embedded tissues are very encouraging.     

A p50 surface antigen restricted to human urinary bladder carcinomas and B lymphocytes

Summary We have previously described the derivation of a monoclonal antibody, S2C6, to a novel 50 Kdalton antigen associated with human urinary bladder carcinoma. No reactions were obtained with carcinomas of unrelated origin or with normal urothelial cells. However, the antibody also reacted with a similar antigen on some cell lines of B lymphocyte origin. Using large panels of target cells we have now shown that this reactivity was entirely restricted to cells of the B lineage within the haematopoietic system. As opposed to its apparent restriction to malignant cells of the urothelium, the S2C6 antigen was expressed by normal B lymphocytes as well as by many malignant B cells (chronic lymphocytic leukaemia, hairy cell leukaemia and immunocytoma). Pre-B cells derived from acute lymphocytic leukaemia and plasma cells from multiple myeloma lacked the antigen. Expression was significantly enhanced on cultured B cells from Burkitt lymphomas and on Epstein-Barr virus-transformed lymphoblastoid cell lines including those of the pre-B phenotype derived from fetal bone marrow. As judged from the molecular size and the distribution pattern displayed by the S2C6 antigen it appears to be distinct from other B cell antigens previously described. A possible relation of the S2C6 antigen to a receptor for B cell growth factors is discussed.     

Lymphoma associated antigen (LAA)--a unique biomarker for lymphomas.

A lymphoma associated antigen (LAA) isolated from pooled lymph nodes of confirmed Hodgkin's and non-Hodgkin's lymphomas has been purified and characterized. Using a xenogenic rabbit anti-serum, enzyme-linked immunosorbent assay (ELISA) and RIA were developed for LAA. LAA was detected in the sera of all confirmed lymphomas, the test being negative for normals, for patients with benign lymphadenitis and various other types of cancers. Except for a very few false positive results, no false negative was observed. LAA was identified in urine, CSF, saliva and gastric juice of a few lymphoma patients, and the test proved to be of diagnostic potential, as for a few patients it had a lead time of a few months over the histological diagnosis. In order to render the LAA test more precise and specific, monoclonal antibodies were generated by both in vitro and in vivo immunization procedures. Seven monoclonals were generated, viz. 7D6, 7D2, 7G2, 7C5, 6G2, 23B7 and 23G11, which exhibited cytoplasmic staining of frozen sections of malignant lymphoid tissues of B cell derived non-Hodgkin's lymphomas. Two of these monoclonal antibodies, 7D6 and 23B7, revealed strong cytoplasmic staining of frozen sections, impression smears and cytospin specimens of B cell non-Hodgkin's lymphomas. The reactivity was very weak or negative for T cell lymphomas. The test was negative for Hodgkin's disease and controls. These results were confirmed by dot blotting, immunoprecipitation and immunofluorescence study. By ELISA with a sensitivity of 15 ng/ml, serum LAA levels for lymphomas were in the range 72-1250 ng/ml. LAA could not be detected in the sera of normals and controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histopathologic Technique for the Morphological Examination of Fixed or Frozen Solid Tumor Cells in Soft Agar

Two simple techniques are described for preparing sections from soft agar colony cultures of tumor cells. Tumor cells grown in soft agar can be frozen, sectioned, and stained and/or fixed in formalin, embedded in paraffin, sectioned, mounted on glass slides, and stained. The methods are simple and reproducible. These cells can be stained with various stains and the staining quality is excellent. The paraffin blocks and microscope slides can be stored for permanent record. The use of these techniques should provide better understanding of the histomorphologic characteristics of neoplastic cells which grow in soft agar and should expand and refine prognosis and diagnosis of malignant tumors.     

Morphometry in evaluating large cell malignant lymphomas in lymph node biopsies

The values of some morphometric cell parameters were analysed in 12 cases of large cell non-Hodgkin's lymphoma (centroblastic and immunoblastic lymphoma and histiocytic sarcomas) in order to test the utility of the morphometric method as an additional element to the histologic diagnosis and to its further accuracy. The nuclear and nucleolar area, the number of nucleoli per nuclear cross section and the nucleus to nucleolus ratio were investigated. The morphometric data were obtained on histograms using the Giemsa stained sections. Mean values and standard deviations were calculated and statistically analyzed. Statistical evaluation of data showed significant differences in the three groups. Our results proved the usefulness of the morphometric method as a helpful technique of differentiating the large cell malignant lymphomas.     

Fine Needle Aspiration Cytology of High Grade T-Cell Lymphomas In Human T-Lymphotropic Virus Type 1 Carriers

We studied the fine needle aspiration (FNA) cytology of high grade peripheral T-cell lymphomas from eight human T-lymphotropic virus-1 (HTLV-1) positive patients. FNA smears from seven lymphomas showed a distinctive cytologic pattern with a dominance of rounded cells with irregular nuclei and a moderately basophilic cytoplasm. Irregular cells with a pale abundant cytoplasm were present in varying amounts. Some smears contained a few giant cells with cerebriform nuclei. In addition, plasma cells and eosinophils were found. Epithelioid cells were an inconstant finding. On histology these seven lymphomas were assigned to the pleomorphic medium-large cell subtype and all but one were of T-helper phenotype with rearrangements of the T-cell receptor. FNA smears from a lymph node in a patient with a previous histological diagnosis of lymphomatoid papulosis of the gingiva showed a monotonous pattern of large immunoblastic cells with some binucleated variants consistent with a diagnosis of high grade immunoblastic lymphoma, which was confirmed histologically. Our results show that peripheral T-cell lymphomas from HTLV-1 positive patients have cytological patterns which are distinctive enough to allow a conclusive diagnosis of high grade T-cell lymphoma. However, we do not think that the cytology of HTLV-1 positive lymphomas can be differentiated from that of virus-unrelated high grade T-cell lymphomas.     

Effects of tissue processing techniques in acoustical (1.2 GHz) and light microscopy.

In this study the influence of various tissue processing and staining techniques on the acoustical properties of liver tissue was investigated. A qualitative study was performed using ultrasound attenuation as the imaged parameter of a combined optical/acoustical microscope with a 1.2 GHz transducer. Images were made of three sets of adjacent liver sections (6 microns in thickness) which were prepared in ten different ways: fixed by alcohol or formalin; stained by hematoxylin-eosin (HE), toluidine blue (TB) or non-stained; sectioned by a cryostat or by a paraffin microtome. It was concluded that the images obtained from cryostat sections were of much higher quality than those from paraffin sections. Images obtained from sections that were sectioned while embedded in paraffin displayed no detail at all. No consistent effect was noticed with respect to staining by HE or TB. Alcohol fixed sections gave more detailed images than formalin fixed sections. Formalin fixation in combination with cryostat sectioning yielded many cytoplasmic vacuoles.     

Fine needle aspiration cytology of palatine tonsils: a study of 112 consecutive adult tonsillectomies

M. J. Ashraf, N. Azarpira, B. Nowroozizadeh, M. Shishegar, B. Khademi, A. Faramarzi, S. B. Hashemi, A. Hakimzadeh and E. Abedi
Fine needle aspiration cytology of palatine tonsils: a study of 112 consecutive adult tonsillectomies Objective: To study fine needle aspiration cytology (FNAC) findings of tonsillar lesions with histological controls and to assess its role in the diagnostic evaluation of tonsillectomy specimens. Methods: This study consisted of 112 cases that required tonsillectomy, comprising 55 (49.1%) men and 57 (50.9%) women. The ages ranged between 20 and 62 years. The clinical diagnosis in 101 cases was chronic tonsillitis, whereas 11 were suspected of neoplasia. FNAC was performed before tonsillectomy under general or local anaesthesia or on fresh specimens using a 21‐G needle. The smears were stained using Wright–Giemsa and Papanicolaou methods. Histological examination was carried out on surgical specimens of all cases and, when required, immunohistochemistry was performed on histological sections. The diagnostic outcomes between FNAC and surgical biopsy were compared. Results: In this study, 106 cases were diagnosed as chronic tonsillitis/follicular hyperplasia, four cases as non‐Hodgkin’s lymphoma, one as Hodgkin’s lymphoma and one as monophasic synovial sarcoma. All malignant cases were diagnosed by FNAC, but synovial sarcoma was incorrectly diagnosed as squamous cell carcinoma. Five cases clinically suspected of neoplasia were correctly diagnosed as chronic tonsillitis on cytology. Conclusion: Tonsillar aspiration is a safe procedure and is useful in the evaluation of tonsillectomy specimens. However, ancillary tests on cytological material are often needed when neoplasia is suspected and would help clinical management and allow histological examination of cases diagnosed cytologically as lymphoma.     

Reliable LC3 and p62 Autophagy Marker Detection in Formalin Fixed Paraffin Embedded Human Tissue by Immunohistochemistry

Autophagy assures cellular homeostasis, and gains increasing importance in cancer, where it impacts on carcinogenesis, propagation of the malignant phenotype and development of resistance. To date, its tissue-based analysis by immunohistochemistry remains poorly standardized. Here we show the feasibility of specifically and reliably assessing the autophagy markers LC3B and p62 (SQSTM1) in formalin fixed and paraffin embedded human tissue by immunohistochemistry. Preceding functional experiments consisted of depleting LC3B and p62 in H1299 lung cancer cells with subsequent induction of autophagy. Western blot and immunofluorescence validated antibody specificity, knockdown efficiency and autophagy induction prior to fixation in formalin and embedding in paraffin. LC3B and p62 antibodies were validated on formalin fixed and paraffin embedded cell pellets of treated and control cells and finally applied on a tissue microarray with 80 human malignant and nonneoplastic lung and stomach formalin fixed and paraffin embedded tissue samples. Dot-like staining of various degrees was observed in cell pellets and 18/40 (LC3B) and 22/40 (p62) tumors, respectively. Seventeen tumors were double positive for LC3B and p62. P62 displayed additional significant cytoplasmic and nuclear staining of unknown significance. Interobserver-agreement for grading of staining intensities and patterns was substantial to excellent (kappa values 0.60-0.83). In summary, we present a specific and reliable IHC staining of LC3B and p62 on formalin fixed and paraffin embedded human tissue. Our presented protocol is designed to aid reliable investigation of dysregulated autophagy in solid tumors and may be used on large tissue collectives.Key words: Immunohistochemistry, immunofluorescence, formalin fixed paraffin embedded tissue, LC3B, p62, cancer