Correlation between PCNA and AgNOR scores in non-Hodgkin's lymphomas using sequential staining technique.

AIMS: To investigate the relation between the numbers of interphase silver stained nucleolar organiser regions (AgNORs) and immunolabelling with the monoclonal antibody PC10, which demonstrates proliferating nuclei by reacting with proliferating cell nuclear antigen (PCNA). METHODS: The established sequential technique for the demonstration of interphase AgNORs and of antigens in paraffin wax sections was applied to a small series of non-Hodgkin's lymphomas (NHL) of both low and high grade type, together with specimens of palatine tonsil. The numbers of PCNA positive cells were counted in all specimens; in the tonsils the cells were counted in both follicle centres and in the interfollicular areas. The numbers of AgNORs in both PCNA positive and PCNA negative nuclei were then counted. RESULTS: Lower numbers of PCNA positive cells were found in the low grade than the high grade NHL (18-28.4% and 34.2-51.3%, respectively). This was reflected in the two areas of the palatine tonsils, the counts being higher in the follicle centre nuclei than in those in the interfollicular compartments. In general, the numbers of AgNORs were higher in the PCNA positive nuclei than in those lacking the antigen; however, a consistent finding was that relatively high AgNOR scores were observed in PCNA negative nuclei, especially in the high grade lymphomas. In the tonsils, however, the AgNOR counts were much lower in the nuclei lacking PCNA than in those containing it. CONCLUSIONS: The results obtained in the lymphomas were rather unexpected as, in general, previous studies have shown a close direct or indirect relation between AgNOR scores and proliferating cell counts. An explanation for these findings may be that the argyrophil proteins associated with proliferating cells remain in the nucleus in detectable form longer than the PCNA antigen, at least in neoplastic tissue.     

Study of vimentin expression in non-Hodgkin's lymphoma using paraffin sections.

Human non-Hodgkin's lymphomas were studied by means of an avidin-biotin complex immunoperoxidase method using several monoclonal antibodies against the intermediate filament protein, vimentin. The study cases were 61 B-cell lymphomas (including 2 plasmacytomas) and 30 T-cell lymphomas (including 8 cases of mycosis fungoides). Twelve of the 61 B-cell lymphomas were positive for vimentin, and were composed of extrafollicular-center cells such as immunoblastic and plasmacytoid cells. On the other hand, lymphomas of follicular center cell origin were negative for vimentin. All cases of T-cell lymphoma except for 14 (all of 9 AILD-type lymphomas, all of 4 lymphoblastic lymphomas and one diffuse mixed small/large lymphoma) were positive for vimentin. Although vimentin expression appeared to be influenced by various conditions such as the proportion of T- and B-cell subsets, or B-cell proliferation rate, follicular center cells were constantly negative for vimentin.     

Demonstration of light chain monotypia in B cell non-Hodgkin's lymphomas using unfixed freeze-dried and formalin-fixed trypsinised paraffin sections.

Immunohistological light chain analysis was carried out in 55 patients with non-Hodgkin's lymphoma of B cell origin. Unfixed freeze-dried paraffin sections were used to detect surface Ig and formaldehyde-fixed trypsinised sections to detect cytoplasmic Ig. Cytoplasmic Ig was seen inconstantly in freeze-dried paraffin sections. There was complete agreement with regard to the type of light chain between freeze-dried and formaldehyde-fixed trypsinised sections. Immunohistology showed the monotypic immunoglobulin light chains in 83% of cases when unfixed freeze-dried paraffin sections were used for sIg demonstration while only in 45% of cases when formaldehyde-fixed material was used for cIg detection. The efficiency of sIg demonstration in unfixed freeze-dried paraffin sections was comparable with that based on unfixed cryostat sections or on cell suspensions when the lymphocytic lymphoma/CLL group was excluded from the evaluation. The relatively low frequency of monotypic sIg positivity obtained in this group (12/19) is due to the low density of surface Ig in CLL lymphocytes.     

Immunohistochemical staining of non-Hodgkin's lymphoma in paraffin sections using the MB1 and MT1 monoclonal antibodies

We have performed a single blind trial to assess the value of the monoclonal antibodies MB1 and MT1 in lymphoma classification. Sixty cases of non-Hodgkin's lymphoma (NHL) were stained with MB1 and MT1 using an indirect immunoperoxidase technique in paraffin sections. The majority of B tumours (27/33) stained with MB1, and most of the T tumours (24/27) stained with MT1. The MB1 antibody often produced rather weak staining but it was apparently highly specific for B cells, with only three (3/27) of the T tumours (two cases of 'malignant histiocytosis' of the intestine (MHI) and one pleomorphic T-cell lymphoma) displaying 'false' positivity. The MT1 antibody generally produced very strong staining, but it was not very selective, with 14/33 of the B lymphomas displaying 'false' positivity. the cross-reactivity observed in 17 cases led to only three misdiagnoses, two B tumours being designated as T lymphomas and one T tumour being designated as a B lymphoma. In a few cases (7/17), dual staining with both antibodies precluded firm diagnosis. In other cases (6/17), classification was possible despite some of the tumour cells showing dual staining. The seventeenth case was a plasmacytoma displaying MT1 positivity only. While the monoclonal antibodies MB1 and MT1 are of use in classifying lymphomas in paraffin section, they are not entirely lineage-specific, and the uncritical use of these two reagents alone may give rise to misdiagnosis; the use of a panel of monoclonal antibodies may yield more accurate results. As with any immunohistochemical marker, their limitations should be recognized; interpretation must be judicious and always in the context of the histological appearances.     

Improved silver staining of nucleolar organiser regions in paraffin wax sections using an inverted incubation technique.

A new simple modification to the silver staining of nucleolar organiser regions (AgNORs) was devised which, by performing the incubation with the slide inverted, results in minimal undesirable background staining, a persistent problem. Inverted incubation is facilitated by the use of a commercially available plastic coverplate. This technique has several additional advantages over other published staining protocols. In particular, the method is straightforward, fast, and maintains a high degree of contrast between the background and the AgNORs.     

My4 antibody staining of non-Hodgkin's lymphomas.

The My4 antibody, one of a number of monoclonal antibodies that react with the CD14 antigen, was originally reported to weakly stain monocytes, macrophages, and granulocytes. However, recent studies have shown that the My4 antibody also stains normal peripheral blood B lymphocytes and some subtypes of B-cell non-Hodgkin's lymphoma. Thus, the authors have studied a large series of non-Hodgkin's lymphomas stained with the My4 antibody. In frozen sections of reactive lymph node biopsy specimens, the My4 antibody strongly stained mantle zone B lymphocytes and weakly reacted with dendritic reticulum cells and histiocytes. In a series of 245 non-Hodgkin's lymphomas, the My4 antibody stained 111 (45%) cases: 108 of 189 (57%) B-cell lymphomas, 3 of 50 (6%) T-cell lymphomas, and 0 of 6 null cell lymphomas. My4-positive B-cell lymphomas occurred in all histologic subtypes with the exception of small noncleaved cell lymphomas. Follicular lymphomas were most often My4 positive (82%). My4 antibody staining showed no correlation with Working Formulation grade. All three My4-positive T-cell lymphomas had a mature T-cell phenotype. Seventy-six of the 111 (68%) My4-positive lymphomas were also analyzed with at least one other anti-CD14 antibody, either Mo2 and/or Leu-M3. In all cases the antigens that react with Mo2 and Leu-M3 were not expressed. Thus, the staining of reactive and neoplastic B cells by My4 appears to be unique to this antibody and is not a feature of all anti-CD14 antibodies.     

Detection of cell surface antigens in cryostat sections with immunogold-silver staining

Immunogold-silver staining was used for the detection of lymphocyte cell surface antigens in cryostat sections of lymphoid tissues. The sections were incubated with monoclonal mouse antibodies and then with colloidal gold-labeled goat anti-mouse antibodies. They were then immersed in a physical developer, counterstained, and mounted. In light microscopy, the tissue architecture was well preserved, and a dark labeling was seen on the positive cells. Optimal labeling conditions were determined. The distribution of the lymphocyte subsets, as defined by a panel of monoclonal antibodies in tonsil and reactive lymph nodes, was similar to that found with a biotin-avidin-horseradish peroxidase method. The monoclonality of the neoplastic cells in lymph nodes of B-cell non-Hodgkin's lymphomas clearly could be demonstrated. The sensitivity of the technic was comparable with that of the biotin-avidin-horseradish peroxidase labeling method. In addition, immunogold-silver labeling was combined with acid phosphatase cytochemistry.     

New technique for differential staining of myelinated fibers and nerve cells on paraffin sections

A simple, rapid method for the differential staining of myelinated nerve fibers and nerve cell bodies, applicable to sections of central nervous system pieces embedded in paraffin, is described. Experimental material fixed by perfusion with mixed aldehydes or necropsy material fixed in formaldehyde can be used. Constant and homogeneous results are obtained with this technique, and the most important characteristic is the absence of differentiation in either of the steps: staining of myelinated fibers and staining of nerve cell bodies. Sections 15 microns thick were attached to slides, dewaxed, and hydrated. After hydration, sections are mordanted (30 min) in 2.5% iron alum (SO4)2FeNH4, and rinsed (1 min) in distilled water. Staining is for 180 min in the following solution: 5 ml freshly made 20% alcoholic hematoxylin diluted with 25 ml of distilled water and 25 ml of absolute ethanol to which 10 ml of 1% Li2CO3 is added. The sections are washed in distilled water (5 min) and stained during 5 min in the following solution: 0.2% pyronine, 20% formaldehyde in distilled water. The sections are dehydrated through 96% and absolute ethanol, cleared in eucalyptol, and mounted in Eukitt. Myelinated fibers appear dark blue, whereas nerve cell bodies are stained red and the cell nucleoli dark blue. This procedure provides an adequate contrast for observation and photography.     

Orcein staining of hepatitis B antigen in paraffin sections of liver biopsies.

Liver biopsies from 97 hepatitis B antigen (HBsAG)-positive patients were stained by a modified orcein method described by Shikata et al (1974) in order to detect the antigen in liver tissue. The results were consitently negative in acute hepatitis, but positive in nearly two-thirds of biopsies from 53 patients with chronic liver disease. The distribution of positive staining was frequently irregular so that there is a problem of sampling error in needle biopsies. The deposits were seen in the cytoplasm of liver cells and occasionally in Kupffer cells, but never in nuclei. There was an inverse relationship between staining and parenchymal necrosis. Biopsies from asymptomatic HB(s)Ag carriers were often strongly positive, as were "ground-glass" hepatocytes in carriers and patients with chronic liver disease. The mechanism of staining is unclear but may be related to the presence of disulphide bonds in (HBsAG. The technique is simple and of use both in fresh and stored material.     

Demonstration of Lipiodol in paraffin sections using a modified silver impregnation technique

To demonstrate postangiographic Lipiodol (LIP) in hepatocellular carcinoma (HCC) in paraffin sections, direct impregnation of formalin-fixed tissue blocks with silver nitrate (AgNO3) was followed by routine processing. LIP appeared as black globules in the sinusoids. Ninety-four tissue blocks from 13 postangiographic LIP HCCs and 69 from 8 non-LIP HCCs and 4 fatty livers were studied. Seventy-two of 73 negative controls and all positive blocks as seen on soft tissue radiographs (STRs) were correctly coded (specificity 98.6%, sensitivity 100%). Twenty-six of the 44 LIP-negative areas on STRs from LIP cases contained scanty globules of less than 10 microns in diameter. Fatty change gave no positive readings. Thus, modified AgNO3 impregnation is a simple, accurate means of detecting LIP in high-quality paraffin sections suitable for tumor diagnosis and, if applied to postangiographic LIP, ultrasonographically guided liver biopsy, can verify that a biopsy has reached a suspected tumor focus.     

Immunocytochemical staining of progesterone receptor in paraffin sections of human breast cancers.

Monoclonal antibodies (mAB) against progesterone receptor (PR) and the peroxidase-antiperoxidase (PAP) method to visualize PR in paraffin sections from 68 human breast cancers were used. Ten mAB, which recognize human PR on frozen sections, were tested. Six could detect PR in paraffin sections, with Li 417, LET 456, and LET 126 giving the best results. LET 126 antibody was used for most further studies. The effects of fixation with picric acid-formaldehyde (PAF), buffered-formalin, or with Bouin solution were investigated; all fixation methods allowed PR immunolabeling, although PAF or buffered formalin usually gave the best results. Positive staining was seen in the nucleus of carcinoma cells. Variations in intensity and extent of immunoreactivity were observed in all sections and among different regions of the same specimen. These were probably related to the heterogeneity of the tumor cell population. Results were compared with the PR content in the respective tumor tissues, determined by steroid-binding assay, and with immunocytochemistry on frozen sections. It was shown that there were correlations between the immunocytochemical staining (positive or negative) and the steroid binding assay (80%) and between the immunocytochemical staining on paraffin sections and on frozen sections (78%). Weaker intensity and fewer number of PR-positive cells were found for paraffin-embedded tumors. Estrogen receptors were also detected on adjacent sections from the same paraffin-embedded tissues by use of monoclonal anti-ER antibodies (ERICA-kit[Abbott Laboratories, Chicago, IL]) and DNase pretreatment. In conclusion, this immunocytochemical method for detection of PR and ER on paraffin sections offers an alternative to frozen tissue. It allows histologic and immunocytochemical studies on the same sample and retrospective studies on stored tissue blocks.     

Application of immunofluorescent staining on paraffin sections improved by trypsin digestion.

The purpose of this study was to explore a method by which an improved immunofluorescent staining can be applied to formalin-fixed paraffin sections to demonstrate cellular or tissue deposits of immunoglobulins, complement and fibrin, and to demonstrate alpha-1-antitrypsin storage and hepatitis B antigens in liver, toxoplasma in heart, and carcinoembryonic antigens in colonic cancer. It was shown that immunohistochemical demonstration for the above mentioned antigens, but not for complement, was feasible. The paraffin sections were first treated with trypsin and the indirect staining method was used. The trypsin treatment was found to decrease the nonspecific background fluorescence through digestion of the tissue. It probably also unmasked the immunoreactive sites of viral antigens and alpha-1-antitrypsin. In general, a 2-hour digestion was satisfactory for the types of tissues examined in this study, and an optimal period of digestion could be sought to obtain the best result for a specific antigen. This method may be a useful adjuvant to histopathologic study, in which a retrospective immunohistochemical examination may be desirable.     

Immunophenotyping of Nigerian cases of non-Hodgkin''s lymphomas on paraffin sections

One hundred cases of routinely fixed and processed non-Hodgkin's lymphoma from Nigeria were immunostained with a small panel of monoclonal antibodies against B-, T- and macrophage antigens. The aims of the study were to assess the suitability of stored material from a country like Nigeria for immunohistochemical examination and the ability of the antibody panel to evaluate the distribution of B- and T-cell neoplasms. Eighty-seven of the 100 cases gave interpretable immunostaining, with 75 being B-cell and 12 T-cell neoplasms. Eighty-seven of the 100 cases gave interpretable immunostaining, with 75 being B-cell and 12 T-cell neoplasms. There were no tumours of macrophage lineage. Four cases gave satisfactory staining of reactive lymphoid cells but no reactivity with malignant cells and thus were not phenotyped. The remaining nine cases gave no staining of neoplastic or reactive cells, suggesting that they were unsuitable for immunohistochemical study, presumably because of inappropriate fixation and handling. We concluded that a panel of three monoclonal antibodies is suitable for routine immunostaining of conventionally fixed and processed blocks in Third World countries and will give diagnostically useful information in approximately 95% of cases.     

An immunoperoxidase technique for demonstrating membrane localized HBsAg in paraffin sections of liver biopsies

An improved unlabelled antibody enzyme (PAP) method to demonstrate the localization of Hepatitis B surface Antigen (HBsAg) in liver cell membranes of paraffin embedded liver tissue, fixed in Bouin's solution, is described.     

Immunophenotyping of non-Hodgkin's lymphomas in paraffin-embedded tissue sections. A comparison with genotypic analysis.

Several monoclonal antibodies (MoAbs) are now available for immunophenotyping non-Hodgkin's lymphomas (NHLs) in paraffin-embedded tissue sections. To determine the reliability of these reagents in predicting the genotype, 44 cases of NHL were studied with the alkaline phosphatase-anti-alkaline phosphatase technique with the use of the following MoAbs: leukocyte common antigen (CD45), Mac 387, L26, 4KB5, MB1, MB2, LN2, UCHL1, MT1, and MT2. The lineage of the neoplastic cells was determined in all cases by gene rearrangement studies for immunoglobulin heavy chain and for the T-cell receptor beta-chain. Genotypic results showed B-cell lineage in 33 cases (75%), T-cell lineage in 6 cases (14%), and mixed or undetermined lineage in 5 cases (11%). A concordance of lineage assignment by paraffin section immunophenotyping with gene rearrangement studies was observed in 37 of 39 (95%) lymphomas with an unequivocally defined genotype. MoAb L26 was the most sensitive in detecting B-cell genotype; MoAbs MT1 and UCHL1 were the most sensitive and specific, respectively, in detecting T-cell genotype. The authors conclude that lineage assignment of NHLs in paraffin sections is reflective of the corresponding genotype when an appropriate panel of MoAbs is used.