Tissue Imprints Research Articles

The value of E‐cadherin/ β‐catenin expression in imprints of NCSLC: Relationship with clinicopathological factors

Metastasis is specific for malignant tumors and its control is one of the most important problems in the design of therapies for cancer patients. Loss or reduction of E-cadherin expression and/or beta-catenin expression plays a casual role in tumor progression and metastasis and is associated with poor prognosis. The purpose of the study is to investigate the expression of E-cadherin and beta-catenin and their significance as independent prognostic markers in imprints of resected nonsmall cell lung cancer (NSCLC). Imprint smears from 70 patients who underwent surgical lung resection for primary carcinoma were studied. As control group was used imprints of physiological tissues. Histologically 47 (67.1%) of the tumors were squamous cell carcinomas and 23 (32.9%) were adenocarcinomas. Tumors stage was I in 29 (41.4%), II in 13 (18.6%), III in 24 (34.3%) and IV in 4 (5.7%). Positive expression for E-cadherin was observed in 44.29% of malignant smears vs 85.71% for control group (P = 0.011). For beta-catenin, positive expression was observed in 42.86% malignant cases vs 85.71% for control group (P = 0.008). Positive expression of E-cadherin and beta-catenin was observed in moderate and well differentiated tumors (P < 0.0001 for both respectively). Positive E-cadherin and beta-catenin expression was observed in 70.6% and 76.5% of the cases with negative lymphnode metastasis (P < 0.0001 for both respectively). There was no statistically significant association between histological type, tumor stage, pleural invasion, tumor size (P > 0.05 for all) and E-cadherin/beta-catenin expression.Reduced E-cadherin or beta-catenin negative expression relates to dedifferentiation and progression of NSCLC.

Evidence for bird‐like air sacs in saurischian dinosaurs

Among extant tetrapods, pneumatic postcranial bones are only present in birds, and they are osteological correlates of the diverticular lungs and pulmonary air sacs. The presence of postcranial pneumaticity in sauropod and theropod dinosaurs suggests that some form of air sac system was also present in the dinosaurian ancestors of birds. In particular, anatomical and evolutionary patterns of pneumatization in nonavian saurischian dinosaurs are diagnostic for specific air sacs, including the cervical, clavicular, and abdominal air sacs. Pneumatic hiatuses are gaps in the pneumatization of the vertebral column and indicate pneumatization from multiple sources. A pneumatic hiatus in Haplocanthosaurus provides additional support for the presence of abdominal air sacs in sauropods. The origins of postcranial pneumaticity in archosaurs are enigmatic because the earliest putative traces of pneumaticity are difficult to distinguish from skeletal imprints of other soft tissues. Nevertheless, several lines of evidence suggest that air sac-driven lung ventilation was primitive for Saurischia.

Evolutionary history of trypanosomes from South American caiman (Caiman yacare) and African crocodiles inferred by phylogenetic analyses using SSU rDNA and gGAPDH genes

In this study, using a combined data set of SSU rDNA and gGAPDH gene sequences, we provide phylogenetic evidence that supports clustering of crocodilian trypanosomes from the Brazilian Caiman yacare (Alligatoridae) and Trypanosoma grayi, a species that circulates between African crocodiles (Crocodilydae) and tsetse flies. In a survey of trypanosomes in Caiman yacare from the Brazilian Pantanal, the prevalence of trypanosome infection was 35% as determined by microhaematocrit and haemoculture, and 9 cultures were obtained. The morphology of trypomastigotes from caiman blood and tissue imprints was compared with those described for other crocodilian trypanosomes. Differences in morphology and growth behaviour of caiman trypanosomes were corroborated by molecular polymorphism that revealed 2 genotypes. Eight isolates were ascribed to genotype Cay01 and 1 to genotype Cay02. Phylogenetic inferences based on concatenated SSU rDNA and gGAPDH sequences showed that caiman isolates are closely related to T. grayi, constituting a well-supported monophyletic assemblage (clade T. grayi). Divergence time estimates based on clade composition, and biogeographical and geological events were used to discuss the relationships between the evolutionary histories of crocodilian trypanosomes and their hosts.

Thiazin red as a neuropathological tool for the rapid diagnosis of Alzheimer’s disease in tissue imprints

In recent years, we have used a variety of tau immunological markers combined with the dye thiazin red (TR), an accurate marker to differentiate the fibrillar from the nonfibrillar state of both amyloid-beta and tau in Alzheimer's disease (AD). In this study, we used TR as a potential diagnostic marker of AD in frozen-thawed (F-T) brain tissue and imprint cytology. Control experiments included the use of Thioflavin-S staining, fixed tissue, and some double-labeled material with TR and selected tau markers, including AT100, MC1, Alz-50, TG-3, Tau-C3, and S396. Our results indicate that TR retains its strong affinity for both tangles and plaques in unfixed F-T tissue and imprint cytology. This information provides a potential use of TR as an accurate diagnostic tool for the rapid postmortem diagnosis of AD neuropathology. This study shows the advantages of TR on cytology mainly because tools for the fast postmortem diagnosis of AD are practically nonexistent. In addition, we observed Tau-C3 immunoreactivity in extracellular tangles, suggesting that the Tau-C3 epitope is characteristically stable. Moreover, this study demonstrates that chemical fixation is not necessarily required for tau immunoreactivity on histological sections.

Rearrangements of MALT1, IgH, FOXP1, BCL3 and PAX5 Are Rare Events in Extranodal Marginal Zone Lymphoma in the UK.

A number of balanced translocations have been described in extranodal marginal zone lymphoma (ENMZL). The t(11;18) and variant t(14;18) which deregulate MALT1 are the commonest reported occurring in up to 40% of patients. The t(3;14) which deregulates FOXP1 is reported to occur in up to 10% of cases whilst the t(1;14) and t(14;19) which deregulate BCL10 and BCL3 respectively have been described in a variable proportion of patients. The purpose of this study was to evaluate the incidence of such abnormalities in the routine diagnostic setting in a geographically defined region of Northern England. We have therefore reviewed a series of 56 consecutive cases of presenting ENMZL, diagnosed between November 2003 and July 2006 in the Yorkshire and Humberside Haematology Network (YHHN), which serves a population of approximately 3.6 million. Diagnoses were made using WHO criteria. Presentation tissue included breast (2), gastric (29), colon (3), Conjuctiva/orbit (3), connective tissue/muscle (3), lung (2), parotid (2), salivary gland (2), thyroid (1), prostate (2), and skin (4). In 2 cases the site was unknown. All cases were investigated for MALT1 rearrangements with an alpha satellite 18 control (VYSIS 32-190055/D18Z1) using interphase FISH at the time of diagnosis. Cases with available biopsy material were further investigated for rearrangements of IgH, (VYSIS 32-191019), PAX5, BCL3 (DAKO Y5413 and Y5411) and FOXP1 (in-house probe). FISH was perormed on thin paraffin sections in 54/56 cases and on fresh tissue imprints in 2 cases. 6/56 (11%) cases demonstrated a rearrangement of MALT1, 5/6 were of gastric origin and 1 colonic. 16/56 (29%) cases demonstrated trisomy 18. 6/44 (14%) cases showed rearrangement of IgH (3/6 were unbalanced rearrangement with loss). In 1 of these cases (gastric) BCL3 was also rearranged, suggesting that this may represent a t(14;19)(q32;q13). 1 additional case (parotid) showed an unbalanced rearrangement of BCL3 with loss of the telomeric portion of the gene, giving a total of 2/30 (7%) cases with BCL3 abberrations. In the latter case no other abnormalities were demonstrated. A further 5 cases were suspicious for IgH rearrangements with a low level of cells (<10%) showing rearrangement. No cases were demonstrated to have rearrangement of PAX5 or FOXP1, but 6/32 (19%) cases showed 3 copies of PAX5 and similarly 7/30 (23%) had extra copies of FOXP1, consistent with complete or partial trisomy of chromosomes 9 and 3.The incidence of MALT1 rearrangements in this study is significicantly lower than most published series. The reasons for this apparrent discrepancy is unclear at present but this study represents a consecutive series of patients presenting in a defined UK cancer network and is therefore free from any referral bias. We similarly failed to demonstrate rearrangements of either FOXp1 or PAX5. Rearrangement of BCL3 was however demonstrated but at low frequency. It is clear that although significant advances have been made in our understanding of the pathobiology of ENMZL the underlying cytogenetic defect remains unknown in the majority of patients.

Use of PCR–RFLP to identify Leishmania species in naturally-infected dogs

Tissue imprints on Giemsa stained slides from dogs were used to investigate the presence of Leishmania amastigotes by either optical microscopy (OM) or Polymerase chain reaction (PCR) detection of DNA. Samples from skin, spleen, lymph node, liver and bone marrow from a Leishmaniasis endemic area dogs where Leishmania ( Leishmania) chagasi and Leishmania ( Viannia) braziliensis are sympatric were studied. Dogs were initially diagnosed by Indirect Immunofluorescence (IIF), as which 39 were IIF positive (≥1:40) and 16 negative. The IIF positive dogs were clinically grouped as symptomatic ( n = 15), oligosymptomatic ( n = 12) and asymptomatic ( n = 12). Although PCR positivity was higher in symptomatic dogs, specially their skin samples, there was no significant difference among clinical groups or organs examined. Ten (62.5%) out of 16 IIF and OM negative animals were positive for PCR in at least one organ. Forty-eight positive PCR amplicons were further submitted to RFLP for Leishmania identification. All dogs were infected with L. ( L.) chagasi except one, infected with L. ( V.) braziliensis. PCR was more efficient than IIF and OM to diagnose canine visceral Leishmaniasis (CVL), regardless of the organ examined and the clinical form present. The use of PCR together with serology helps determining the extension of sub clinical infection in CVL endemic areas and provides a better estimate of the number of dogs to be targeted for control measures. In conclusion, our data reinforce the need for a specific diagnosis of canine infection in areas where diverse Leishmania species are sympatric and demonstrate that PCR–RFLP can be used to identify Leishmania species in dog tissue imprint stained slides.

Stratification of non-small cell lung cancer patients for therapy with epidermal growth factor receptor inhibitors: the EGFR fluorescence in situ hybridization assay

DNA fluorescence in situ hybridization (FISH) technology is used to study chromosomal and genomic changes in fixed cell suspensions and tissue block preparations. The technique is based on specific hybridization of small labeled DNA fragments, the probes, to complementary sequences in a target DNA molecule. Demand for FISH assays in formalin-fixed, paraffin-embedded tissues has been increasing, mainly in conditions in which diagnosis is not achieved in cell smears or tissue imprints, such as solid tumors. Moreover, the development of molecular targeted therapies in oncology has expanded the applicability of tests to predict sensitivity or resistance to these agents. The efficient use of tyrosine kinase inhibitors (TKI) of the epidermal growth factor receptor (EGFR) as therapeutical agents in advanced non-small cell lung cancer (NSCLC) depends on identification of patients likely to show clinical benefit from these specific treatments. The EGFR gene copy number determined by FISH has been demonstrated as an effective predictor of outcome from NSCLC patients to EGFR TKIs; however there are pending challenges for standardization of laboratory procedures and definition of the scoring system. This methodology article focuses on the EGFR FISH assay. It details the scoring system used in the studies conducted at the University of Colorado Cancer Center in which a significant association was found between increased EGFR copy numbers and clinical outcome to TKIs, and proposes interpretative guidelines for molecular stratification of NSCLC patients for TKI therapy.

Tissue Imprints or Primary Cultures

Touch preparations or imprints have been extensively used in cytogenetics to avoid primary cultures, especially when studying solid tumors which are hard to grow in vitro. Interphase nuclei studies by FISH have been validated in several sample types; however, to our knowledge, a comparison between both methods when studying clonality has not yet been published. We have performed a comparative FISH study between touch preparations and cultured cells to assess their reliability when studying the aneuploidy of chromosome Y in mosaicism. Our results in 23 samples indicate that aneuploidy of chromosome Y assessed in cells from tissue cultures versus cells obtained from touch preparations from seminal vesicles of patients with prostate cancer is not comparable. The percentage of aneuploid cells is higher in cultured cells. Attention, therefore, must be paid not to overestimating or underestimating the number of aneuploid cells detected when using interphase FISH studies, especially in solid tumors where clonality is very frequent. Also, according to our results, it is reasonable to extrapolate that when performing interphase nuclei studies in paraffin sections or tissue microarray, and therefore underestimations of aneuploidy could be reported. This might be of special relevance if the aneuploidy detected correlates with the tumor progression or might be used as a prognostic factor.

Numerical Aberrations of Chromosomes 1 and 7 by Fluorescent in Situ Hybridization and DNA Ploidy Analysis in Breast Cancer

The goals of this study were to detect the numerical alterations of chromosomes 1 and 7 in breast cancer and to correlate the findings with DNA ploidy status as well as with parameters of prognostic significance. Fluorescence in situ hybridization (FISH) with centromeric probes for chromosomes 1 and 7 and cellular DNA content measurement by image analysis-based cytophotometry were applied on interface nuclei from fresh tissue imprints of 59 breast ductal carcinomas. Immunohistochemical stainings for estrogen receptor (ER), progesterone receptor (PR), HER-2, p53, and Ki67 were performed on paraffin tumor sections. The correlation between DNA ploidy and chromosomal aberrations revealed a significant association between aneuploidy and aneusomy for both chromosomes 1 (p=0.002) and 7 (p=0.00001), however, a number of diploid tumors were found to be aneusomic, especially for chromosome 1. Chromosome 7 polysomy was significantly associated with a higher incidence of axillary lymph node metastasis (p=0.05), poorly differentiated (grade III) tumors (p=0.03), negative ER and PR status (p=0.02 and 0.001, respectively), as well as p53 protein expression (p=0.05) and a higher Ki67 labeling index (p=0.004). Chromosome 1 aneusomy was only related with HER-2 protein overexpression (p=0.05). No association between chromosome alterations and tumor size was detected. In conclusion, the results of our study indicate that the detection of numerical aberrations of chromosomes 1 and 7 by FISH seems to be more sensitive than DNA ploidy status for the evaluation of abnormal cellular DNA and chromosome 7 aneusomy characterizes tumors with aggressive features and therefore might be a useful predictor of unfavorable biological behavior in breast cancer.

Diagnóstico pós-morte de Aelurostrongylus abstrusus e caracterização morfométrica de ovos e mórulas por meio de histologia e impressão de tecido

Os objetivos deste trabalho foram diagnosticar Aelurostrongylus abstrusus por meio das técnicas de histologia e impressão de tecido pulmonar de gato doméstico e realizar a caracterização morfométrica de ovos e mórulas desse nematódeo. Pela histologia, os ovos (n=100) mediram, em média, 83,4 x 65,5mim, índice morfométrico (IM) 0,8 e as mórulas (n=100) mediram, em média, 45,4 x 31,8mim, IM 0,7. Pela impressão de pulmão, os ovos (n=100) mediram, em média, 105,3 x 93,6mim, IM 0,9, e as mórulas (n=100) mediram, em média, 43,1 x 36,5mim, IM 0,8. A utilização da técnica de impressão de tecido permitiu a observação das mesmas formas parasitárias visualizadas histologicamente. Os resultados sugerem a viabilidade da utilização dessa técnica no diagnóstico pós-morte de A. abstrusus.

Micromanipulation of single cells from tissue imprints is an alternative to laser‐assisted microdissection

The characterization and analysis of single cells by molecular biological methods such as the polymerase chain reaction (PCR) is of increasing interest in biomedical research. Different techniques have been developed to obtain single cells from solid tissue. Currently, the most frequently used technique is laser-assisted microdissection (LAM). However, LAM of tissues cannot exclude contamination of the targeted cells by underlying cell fragments. Moreover, this technique can only be performed if a laser microscope is available. Thus, we developed a method to obtain single cells of fresh solid tissue by the simple technique of tissue imprints. After immunostaining of the imprints, single cells were transferred to a reaction tube using a 27-gauge needle guided by a mechanical micromanipulator. Consequently, we used these cells in a single cell PCR.

Lymphoma associated chromosomal abnormalities can easily be detected by FISH on tissue imprints. An underused diagnostic alternative

Background: Fluorescence in situ hybridisation (FISH) is useful for detecting specific chromosomal abnormalities in various tumours. In lymphomas, diagnosis is frequently made using paraffin wax embedded tissue. However, FISH performed...

Occurrence of stone fruit viroids in Bosnia and Herzegovina.

Tissue-imprint hybridization (TIH) assays were used to determine the occurrence and incidence of Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd) in stone fruit trees in Bosnia and Herzegovina. Our collections included trees of plum, peach, cherries, apricot, myrobalan and blackthorn from 33 commercial orchards and 2 nurseries, in the areas of Banja Luka, Gradacac, Sarajevo and Mostar. Of the 410 trees assayed, 44 (11%) tested positive by TIH assays. PLMVd was detected in 39 peach trees, including two old (seed grown) vineyard peach trees (Prunus persica subsp. vulgaris). Tests for HSVd were positive in 3 apricot and 2 plum trees. PLMVd was widely distributed throughout the country. In contrast, HSVd was found only in the northern part of the country. Both native and imported cultivars of Prunus were infected. This is the first record of PLMVd and HSVd in Bosnia and Herzegovina. In a separate experiment, peach trees with PLMVd were monitored in the autumn, winter and spring seasons, with tissue imprints of leaf petioles, dormant cuttings and forced sprouts from dormant cuttings. Irrespective of the tissues assayed, nearly all samples tested positive for PLMVd.

Fluorescent human lung macrophages analyzed by spectral confocal laser scanning microscopy and multispectral cytometry

Numerous highly fluorescent macrophages (MPhi), designated "smoker cells," exist in the lungs of smokers and subjects who have quit smoking within 5 years. The brightly fluorescent MPhi, however, are not present in the lungs of never smokers. Some investigators have speculated that the intense fluorescence of the MPhi is due to smoke-induced changes in the autofluorescence of naturally occurring (i.e., endogenous) compounds (e.g., NADP). In contrast, other researchers have theorized that the fluorescence is due to the uptake of tobacco smoke particulates (i.e., "tar"). Studies reported herein were undertaken to test the hypothesis that the origin of the MPhi fluorescence could be profiled with the novel technologies afforded by spectral confocal laser scanning microscopy (sCLSM) and multispectral cytometry (MSC). To this end, spectral emissions were obtained by sCLSM of optical sections of live MPhi isolated from fresh surgically excised human lung tissue and in air-dried lung tissue imprints. Confirmation of spectral profiles of these single cell observations was obtained in population studies with the use of high-throughput MSC in which multispectral analyses were performed with three different lasers. Proof of concept experiments demonstrated that relatively nonfluorescent MPhi from the lungs of nonsmokers became fluorescent upon short-term ex vivo exposure to tobacco smoke tar. Summarily, the studies reported herein document that the fluorescence of human lung MPhi is due to tobacco tar.

Lymphoma Associated Chromosomal Abnormalities Can Be Easily Detected by FISH on Tissue Imprints. an Underused Diagnostic Alternative.

Detection of specific chromosomal abnormalities is essential in the diagnosis of several lympholiferative disorders. However, conventional cytogenetic studies are not frequently carried out from biopsies as it is in bone marrow (BM) specimens. On the contrary, FISH is usually performed on paraffin embedded tissue, an alternative with potential technical nuances both in its application and its interpretation. In our experience, FISH on tissue imprints is the ideal alternative to overcome these problems. In the present study, 46 tissue imprints and 17 BM smears from 43 patients with lymphomas were selected to investigate the presence of t(14;18)(q32;q21), t(11;14)(q13;q32), t(8;14)(q24;q32) and t(3;var)(q27;var). Representativity of the samples was assured prior to FISH by rapid May-Grümwald staining (Diff-Quick, QCA, Spain). Twenty imprints from reactive palatine tonsils and adenoids were used as negative controls. FISH was performed with specific dual-color dual-fusion FISH (D-FISH) probes for the first 3 translocations and a dual-color break-apart FISH probe for t(3;var)(q27;var) following the instructions of the probes supplier (Vysis, Inc). All except one sample (a BM smear stored at room temperature over 9 years), rendered satisfactory FISH hybridizations. In any case, all 43 patients could be successfully studied by FISH (the case referred above in which FISH failed in a first attempt was successfully analyzed using a different sample). The results supported the suspected diagnosis of follicular lymphoma in 22 patients, mantle cell lymphoma in 12 patients, large B-cell lymphoma in 5 patients, marginal zone lymphoma in 2 patients and B chronic lymphocytic leukemia in 2 patients. In one case, the observation of t(11;14) by FISH allowed to reclassify the case from follicular to mantle cell lymphoma and also to demonstrate clonal evolution by studying sequential tissue imprints stored for more than 6 years. Negative results also aided in the assignment of patients to proper diagnostic categories. FISH performed on tissue imprints and BM smears constitutes an optimal strategy for retrospective and prospective investigation of chromosomal abnormalities in lymphomas. Tissue imprints and BM smears conventionally stored at room temperature even for long periods of time can be safely used and sent by ordinary mail without special considerations for this purpose. Based on our experience we recommend to perform and routinely store tissue imprints whenever fresh tissue is available.

Comparison of light microscopic techniques for the diagnosis of the infection of the European flat oyster Ostrea edulis by the protozoan Bonamia ostreae

A comparison among various histological techniques for the detection of the parasite Bonamia ostreae in oysters Ostrea edulis was performed to evaluate their sensitivity and suitability for different purposes. The comparison involved examination of histological sections, tissue imprints from gills, digestive gland, gonad and heart, and haemolymph cell monolayers, prepared through various protocols. Every technique produced some false negative. The haemolymph cell monolayers were more sensitive than tissue imprints and histological sections. Heart imprints provided the highest sensitivity among tissue imprints. Examination of histological sections was among the least sensitive techniques. Four procedures for estimation of infection intensity were compared. Some differences in accuracy for the estimation of infection intensity between haemolymph cell monolayers and histological sections (HS) were detected: there was a very good agreement when the infection appeared low or heavy in HS but it was not so good in the remaining cases. The results suggest the need for a critical review of the recommendations of the “Office Internationale des Epizooties” and the European Union for diagnosis of bonamiosis.

Haploinsufficiency of Anx7 tumor suppressor gene and consequent genomic instability promotes tumorigenesis in the Anx7(+/-) mouse.

Annexin 7 (ANX7) acts as a tumor suppressor gene in prostate cancer, where loss of heterozygosity and reduction of ANX7 protein expression is associated with aggressive metastatic tumors. To investigate the mechanism by which this gene controls tumor development, we have developed an Anx7(+/-) knockout mouse. As hypothesized, the Anx7(+/-) mouse has a cancer-prone phenotype. The emerging tumors express low levels of Anx7 protein. Nonetheless, the wild-type Anx7 allele is detectable in laser-capture microdissection-derived tumor tissue cells. Genome array analysis of hepatocellular carcinoma tissue indicates that the Anx7(+/-) genotype is accompanied by profound reductions of expression of several other tumor suppressor genes, DNA repair genes, and apoptosis-related genes. In situ analysis by tissue imprinting from chromosomes in the primary tumor and spectral karyotyping analysis of derived cell lines identify chromosomal instability and clonal chromosomal aberrations. Furthermore, whereas 23% of the mutant mice develop spontaneous neoplasms, all mice exhibit growth anomalies, including gender-specific gigantism and organomegaly. We conclude that haploinsufficiency of Anx7 expression appears to drive disease progression to cancer because of genomic instability through a discrete signaling pathway involving other tumor suppressor genes, DNA-repair genes, and apoptosis-related genes.

Analysis of Tissue Imprints by Scanning Laser Cytometry

The analysis of DNA content and simultaneous surface or intracellular antigen expression is described for laser scanning cytometry. Measurements can be performed on microscope slides prepared immediately after dissection of native surgical specimens. These protocols will be useful for both diagnostic pathology and cell biology laboratories.

Sensitive Detection of Artichoke Latent Virus in Globe Artichoke Field Samples by One‐step RT‐PCR or Tissue Imprint Hybridization

Abstract Artichoke latent virus (ArLV) is one of the most economically important and widely distributed viruses of artichoke in the Mediterranean area. The availability of a sensitive and reliable diagnostic method is a prerequisite for evaluating the sanitary status of globe artichoke plants. We report here two molecular methods for the detection of ArLV. The first consists of RNA extraction from infected leaf tissue using a commercial kit, followed by a one‐step reverse transcription polymerase chain reaction (RT‐PCR) protocol, using primers designed in this investigation. The second is based on the tissue imprint hybridization technique, using a denatured digoxigenin‐labelled ArLV DNA probe. Both methods were effective in detecting ArLV in late globe artichoke plant samples.

Mantle cell lymphoma: improved diagnostics using a combined approach of immunohistochemistry and identification of t(11;14)(q13;q32) by polymerase chain reaction and fluorescence in situ hybridization

Mantle cell lymphoma (MCL) is a clinicopathological entity characterized by an aggressive clinical course, morphological features, and overexpression of cyclin D1 due to juxtaposition of the bcl-1 locus (and CCND1 gene coding for the cyclin D1) to the IgH gene. This phenomenon is caused by t(11;14)(q13;q32). The morphological diagnosis of MCL may pose difficulties. Ancillary methods are available to support the diagnosis. We studied a group of 32 patients with MCL; 24 men and 8 women. The median age at the diagnosis was 64 years. We characterized the investigated group by histology, and to analyze the immunohistochemical (IHC) profile we used a panel of antibodies including anti-cyclin D1. Polymerase chain reaction (PCR) was used to detect the rearrangement of bcl-1/IgH in 26 cases (in 11 patients, the DNA was isolated from frozen tissues or from nucleated cells of bone-marrow aspirate or peripheral blood, in 15 patients we utilized paraffin-embedded material). Dual color fluorescence in situ hybridization (FISH) on interphase nuclei detecting the t(11;14)(q13;q32) was applied in all 32 cases. Cyclin D1 IHC was positive in 29 of 30 cases tested (97%). In six, the result was weak and difficult to rely on to support the diagnosis. PCR revealed the fusion gene in 14 of the 26 cases (54%). The best yield was obtained from fresh and frozen samples (8 of 11 positive). Using FISH, we identified the translocation in all 32 patients, the findings being easily interpretable in 29 patients. In three cases, the intensity of red and green signals was weaker and difficult to read though the co-hybridized signals were identified. The classical pattern of the translocation was observed in 26 patients, while in 3 we found variant patterns suggesting a loss of the V segment of the IgH gene (2x) and a shift in the breakpoint region at chromosome 11 (1x). The diagnosis of MCL should be supported by a complex laboratory approach. Interphase FISH seems a useful complementary method to morphology and IHC. It is applicable to various tissues and cells prepared as tissue imprints or histological sections.