Application of the FICTION technique for the simultaneous detection of immunophenotype and chromosomal abnormalities in routinely fixed, paraffin wax embedded bone marrow trephines.

The use of interphase fluorescence in situ hybridisation (FISH) to study cytogenetic abnormalities in routinely fixed paraffin wax embedded tissue has become commonplace over the past decade. However, very few studies have applied FISH to routinely fixed bone marrow trephines (BMTs). This may be because of the acid based decalcification methods that are commonly used during the processing of BMTs, which may adversely affect the suitability of the sample for FISH analysis. For the first time, this report describes the simultaneous application of FISH and immunofluorescent staining (the FICTION technique) to formalin fixed, EDTA decalcified and paraffin wax embedded BMTs. This technique allows the direct correlation of genetic abnormalities to immunophenotype, and therefore will be particularly useful for the identification of genetic abnormalities in specific tumour cells present in BMTs. The application of this to routine clinical practice will assist diagnosis and the detection of minimal residual disease.

Molecular insight into the diagnosis of lymphoma.

Malignant lymphoma may be very difficult to diagnose with routine histopathological methods because they may recapitulate benign architecture or contain benign infiltrates. The best method of diagnosis is to establish the clonal profile of the lymphocyte population, since a monoclonal proliferation is highly suggestive of neoplasia. By means of a PCR (polymerase chain reaction) method it is possible to detect the immunoglobulin heavy chain (IgH) gene rearrangement and therefore establish the lymphocyte clonality. PCR with primers complementary to relatively conserved regions called frameworks (FR1-FR3) laying among hyper variable regions (CDR1-CDR3) of IgH gene unable us to detect monoclonal versus polyclonal B-cell population. The length of the PCR product with these primers is unique if we deal with a monoclonal population. On the contrary, a polyclonal population gives PCR products of a different size. In this retrospective study we used a semi-nested PCR to analyse 37 paraffin-embedded specimens. All of them had been evaluated previously by pathohistological and immunophenotypic criteria. A number of polyclonal (PBL and tonsils from healthy donors) and monoclonal cells (PBL from CLL patients, Raji cell line) were analyzed as controls. Clonality was successfully determined in all specimens. Our results support the concept that molecular techniques such as PCR provide a helpful approach for detection of monoclonal immunoglobulin rearrangements in malignant lymphoma. This is especially true for border cases, but always in the combination with other pathohistological methods.

Monoclonality in Helicobacter pylori-positive gastric biopsies: an early detection of mucosa-associated lymphoid tissue lymphoma.

Mucosa-associated lymphoid tissue (MALT) is not present in healthy gastric mucosa, but it can develop in sites of long-persisting inflammation and is connected with the development of MALT lymphoma. A monoclonal lymphocyte population is one of the characteristics of such lymphomas. In this study we analyzed gastric biopsies (formalin fixed and paraffin embedded or frozen) in 93 patients with dyspepsia accompanied by Helicobacter pylori infection. We applied PCR and single-cell immunocytochemistry to detect the clonality of the gastric B-cell population. Immunocytochemistry performed on 33 frozen biopsies showed two samples with monoclonal pattern. PCR analysis of immunoglobulin heavy-chain (IgH) gene rearrangements revealed two monoclonal populations out of 161 biopsies from 60 patients. We conclude that PCR analysis was the most sensitive method, which gave us insight into the nature of the earliest stage of MALT lymphoma in gastric biopsies.

Jumping translocations involving 11q in a non-Hodgkin lymphoma.

This paper presents the results of a cytogenetic analysis in an 11-year-old boy with non-Hodgkin lymphoma. The investigation was performed on slides obtained from short-term culture of lymph node cells. The analyses revealed an abnormal clone with loss of Y, gain of an X chromosome, t(3;22), trisomy 11, and three cytogenetically-related subclones with jumping translocations involving 11q13 as the common breakpoint region. This region is an unusual site of chromosome breakage in jumping translocations, and has not been reported thus far. Contrary to most published reports, the jumping translocation in our patient is associated with long survival.

Computer-based teaching of pathology at the Zagreb University School of Medicine.

AIM: To review the experience gained in transferring USA computer-based teaching system of medical school pathology to Croatia. METHODS: Computer-based teaching program of pathology developed at the University of Kansas School of Medicine, Kansas City, Kansas, USA, was transferred to the University of Zagreb School of Medicine, Zagreb, Croatia. The experimental group of 49 students was enrolled into this computer-based program. Their performance was compared with that of 195 classmates enrolled in the standard course. Objective (performance on the examinations) and subjective data (students' interviews and written evaluations of the course) were analyzed. RESULTS: The computer program was operational 5 months from the inception of the transfer. It was well received by the students, even though many initially complained that it required more effort and a continuous commitment. The major problems concerned scheduling, reflecting various requirements i mposed on students by other departments teaching in parallel with the Pathology course. Objective data gathered so far indicate that the students enrolled in the computer-based program took the first midterm examination at a significantly higher rate than the rest of the class (p<0.001), and passed the examination with significantly better grades (p<0.001). CONCLUSION: Computer-based teaching programs can be readily transferred to other countries. Full implementation of the program, however, may require significant changes in the existing curriculum in the medical school to which such a program has been transferred or considerable modifications in the program adopted for transfer. It appears that the students enrolled in the computer-based program perform better than students in the standard pathology course.

The generation and in vivo differentiation of murine embryonal stem cells genetically null for either N-cadherin or N- and P-cadherin.

Many mutations of the murine genome are recessive embryonic lethals precluding phenotype analysis at subsequent stages of development. This is true for embryos genetically lacking either N-cadherin or N- and P-cadherin. To circumvent this, we have generated pluripotent embryonal stem (ES) cells of the same genotype in vitro and differentiated them in vivo in the form of teratomas. All of the ES cells isolated in this study had a normal ES cell morphology in vitro and were able to generate teratomas. Histological analysis revealed that some differentiation and histogenesis had occurred within the teratomas. Epithelial formation was, for example, unaffected in all cadherin null cells. Surprisingly, however, the differentiation of cells lacking both N- and P-cadherin was, in general, even more pronounced both quantitatively and qualitatively. Tumours lacking either N- cadherin or N- and P-cadherin contained more striated muscle (apparently cardiac muscle) than heterozygote controls, and this was most strikingly conspicuous in teratomas from N- and P-cadherin null cells. This more pronounced differentiation was not seen for all tissues, however, as structures with a simple neural tube-like morphology were never found in teratomas lacking both N- and P-cadherin and organoid-like structures were rare in Ncad-/-tissue.

Helicobacter pylori: in vitro induction of resistance to azithromycin.

Helicobacter pylori resistance to macrolides is possibly an important factor for the failure of macrolide therapy for H. pylori infection. The aim of this study was to assess the propensity of H. pylori to develop in vitro resistance to azithromycin. In 73 clinical isolates taken from patients before starting antimicrobial therapy of H. pylori infection, MIC was determined using an agar dilution method (Müller-Hinton agar with 7.5% unlysed horse blood, pH = 7.2, at 35 degrees C, during 72 h in a humid microaerobic atmosphere). Each strain was first cultivated at half minimal inhibitory concentration (MIC) then in doubling concentrations until growth arrest. All experiments for induction of resistance were performed on the same media, incubation temperature, atmosphere and time of MIC determination. MIC interpretative standards for sensitivity, intermediate sensitivity and resistance of H. pylori to azithromycin were < or = 2, 4 and > or = 8 mg/l, respectively. Of 73 strains, 5 died during the experiments, and in the remaining 68 strains, serial passage with increasing azithromycin concentrations resulted in the development of resistance in 19 (26.9%) strains. Two strains had an MIC of 16 mg/l azithromycin. Thirty-three (48.5%) strains kept the same MIC or doubled their MIC, 16 (23.5%) strains had 4- to 16-fold MIC but still remained sensitive, 2 resistant strains had 128-fold MICs and 17 resistant strains had increased their MICs more than 128 times. Seventeen highly resistant strains (MIC > 128 mg/l) were kept frozen at -70 degrees C for 3 months in a brain-heart infusion broth with 15% glycerol. MIC was assessed again to determine the stability of resistance. Eleven strains kept MICs > 128 mg/l, 2 became sensitive and 1 intermediate, but reverted easily, after only 2 passages, to an MIC of > 128 mg/l azithromycin. Although macrolides are very active against H. pylori, the propensity to develop resistance in a high proportion of strains has a clear impact on the choice of the right combinations of macrolides with other agents as well as the dosage of the macrolide antibiotics.

[Castleman's disease]. / Castlemanova bolest.

In this paper we have presented five patients in whom the diagnosis of Castleman's disease was established. Although it was described for the first time in 1956, the etiology of this lymphoproliferative disorder is still unknown, and the life expectancy is uncertain. Lymph nodes and spleen were analysed with the standard histological procedures and hyaline-vascular or plasma type of Castleman's disease was diagnosed. It is known that plasma-cellular type, especially its systemic presentation, can transform into malignant lymphoma or Kaposi sarcoma. Hyaline-vascular type was considered as a localized, benign lesion until malignant proliferation of the stromal cells were found in patients with this disorder. Out of five patients plasma-cellular type was diagnosed in three of them. In two of these three, malignant lymphoma appeared. Out of two patients with hyaline-vascular type one has developed follicular dendritic cell tumor.

Assessment of Prognostic Factors in 102 Patients with Myelodysplastic Syndrome-Osteoblasts are Related to Survival.

In a group of 102 patients with myelodysplastic syndrome an evaluation of bone marrow biopsy was performed in order to assess prognostic impact on survival. The following characteristics were included in the analysis: cellularity, erythropoiesis, granulopoiesis, thrombopoiesis, lymphatic nodules, mastocytes, dyserythropoiesis, dysgranulopoiesis, dysthrombopoiesis, blasts, atypical localisation of immature precursor cells, fibrosis, osteoblasts and osteoclasts on bone surfaces. Data were statistically analysed by stepwise Cox proportional hazards regression. Survival was compared between FAB sub-classes, with better prognosis for RA and RARS patients and poor for CMML patients. Better survival was found in patients without dysgranulopoiesis and in those with osteoblasts on bone surfaces. Agreement with established prognostic factors indicates a representative sample. Relationship of osteoblasts to better survival shows involvement of bone tissue in MDS, suggesting it might be useful to include assessment of bone tissue features in histopathologic evaluation.

Ciliated metaplasia in a patient of Mediterranean origin with gastric adenoma.

We report here the first case of ciliated gastric metaplasia in a Croatian patient. This is also the first case of ciliated metaplasia reported in a patient of Mediterranean descent. Cilia were found in slightly cystically dilated gastric glands underneath a gastric adenoma with severe dysplasia. They were visualized by desmin immunohistochemical stain. Cells that presented with cilia were columnar cells, some of them with vacuolization of the cytoplasm. This case report shows that ciliated metaplasia occurs in patients of Southern European origin.

A role for cadherins in tissue formation.

We have produced null mutant mouse embryonic stem cells for the cell adhesion molecule E-cadherin. Such E-cadherin-/- ES cells are defective in cell aggregation; this defect can be corrected by transfection with cDNA for either E-cadherin or N-cadherin driven by a constitutive promoter. The presence (or absence) of E-cadherin regulates the expression of the transcription factor T-brachyury, indicating that cadherins play a role in linking cell surface receptors and gene expression. Comparative analysis of the parental and the genetically altered ES cell lines was performed to examine cell differentiation and the capability to form organized tissues. While differentiating E-cadherin-/- ES cells are still able to express various early and late differentiation markers, they show a clear-cut deficiency in forming organized structures. This phenotype can be rescued by constitutive expression of E-cadherin, which results exclusively in formation of epithelia. In contrast, rescue transfectants expressing N-cadherin show no epithelial structures, instead forming neuroepithelium and cartilage. These results provide the first evidence that specific cadherins directly stimulate differentiation into certain types of tissues.

[Fatal "overwhelming" strongyloidiasis in an immunosuppressed patient]. / Smrtonosna "preplavljujuca" strongiloidoza u bolesnika sa suprimiranim imunitetom.

The increased use of immunosuppressants in the treatment of malignant and non-malignant diseases in today's medicine has significantly contributed to the increased interest in infections caused by opportunistic microorganisms and rare parasites. A fifty-eight-year old male patient, professor, born in Bosnia, was admitted to the Institute due to poor general condition and decompensated steroid diabetes. He had been under immunosuppressant therapy for the previous 5 weeks. Six months before, he noticed squamous and crusted changes on capilli, and afterwards on his body too. As these changes did not respond to local therapy he was admitted to the Department of Dermatovenereology, Zagreb University School of Medicine. Histologic analysis indicated pemphigus erythematosus. He was treated with immunosuppressants (methylprednisolone + azathioprine). Endoscopic examinations revealed duodenal ulcer, in addition to diabetes which could not be regulated by oral hypoglycemics. He received antiulcerative therapy for ulcer treatment. Several hours upon admission the patient became highly febrile, and vomited a sanguinolent content. In spite of intensive therapy, he became comatose and died 20 hours later. On autopsy, generalized strongyloidosis of the lungs, liver, duodenum and small intestine, and a bleeding duodenal ulcer due to strongyloidosis were found. This review should remind us that hyperinfestation with strongyloides is a rare and severe complication, and could be expected in immunocompromised patients.

AgNORs predictive value of prognosis in non-Hodgkin's lymphoma according to the Kiel classification.

Application of the colloidal silver method for demonstration of argyrophilic proteins of nucleolar organizer regions (AgNORs) in histological sections of non-Hodgkin's lymphomas (NHLs) has been shown to be a helpful method for the discrimination of high- and low-grade NHL. In this study correlation between the mean number of AgNORs and other morphological and clinical parameters with survival in 58 patients with NHL, classified according to the Kiel classification, was investigated. It was shown that the mean number of AgNORs differed between the low- and high-grade group of NHLs (P < 0.001), as did survival (P < 0.0001). The mean number of AgNORs was also highly significant when describing actuarial survival. The value of 2.9 NORs per nuclei was the cut-point for separating the whole group into two subgroups, which differed the most when related to survival of patients with NHL.

[Vaginal mixed tumor or spindle cell epithelioma--a case report]. / Vaginaler Mischtumor oder Spindelzellepitheliom--ein Fallbericht.

A case of mixed tumor of the vagina or spindle cell epithelioma is presented and the literature on this rare type of tumor is reviewed. Immunohistochemical findings suggest an epithelial origin. Follow-up studies indicate benign behavior. However, recurrent tumors were reported suggesting careful follow-up observation after excision of extended primary tumors.

Embryonal rhabdomyosarcoma with 92,XXYY, +dmin and evidence of spontaneous cell fusion.

The results of a cytogenetic analysis in an embryonal rhabdomyosarcoma are presented. A tetraploid karyotype with double minute chromosomes (dmin) was identified using a direct method of tumor tissue treatment. In 5% of the examined cells, evidence of spontaneous cell fusion was observed.

Intensive care of severely wounded military and civilian casualties in Zadar, Croatia.

From August 1, 1991, to May 30, 1992, 148 severely wounded military and civilian casualties with the injury severity score of 3 to 5 were treated in the intensive care unit of the Zadar General Hospital. There were 138 male and 10 female patients; their mean age was 32 years. There were 64 wounded civilians and 84 wounded soldiers. The average evacuation time was 3 hours. Twelve (8%) severely wounded persons died. The cause of death was craniocerebral injury in 7 patients (58%) and hemorrhage in 4 patients (33%). Complications following shock-like acute renal failure, gastrointestinal bleeding, coagulopathy, and hepathopathy developed in 18 wounded persons (12%).

Expression of P-selectin on endothelial cells is upregulated by LPS and TNF-alpha in vivo.

P-selectin is an endothelial cell adhesion molecule which mediates the binding of neutrophils and monocytes. Its appearance at the cell surface can be induced within minutes by histamine and thrombin which rapidly stimulate the transport of P-selectin from intracellular storage granules to the plasma membrane. We have recently found a second regulation mechanism for P-selectin on mouse endothelioma cells. Like E-selectin, P-selectin is also regulated at the level of transcription. Both selectins are induced by LPS or TNF-alpha with a maximal expression level at the cell surface 3-4 h after stimulation. Here, we report that this up-regulation of the synthesis of P-selectin also occurs in vivo in endothelium of the mouse. Analysing brain tissue, which is devoid of constitutive expression of P-selectin, we found that LPS and also TNF-alpha strongly induce the expression of P-selectin on all venular endothelial cells of the leptomeninges and, at a weaker level, on some blood vessels of the brain parenchyma. Induction of P-selectin expression could also be observed in tissues, such as the tongue, where P-selectin is constitutively expressed on small venules but only rarely on larger venules. Strong staining for P-selectin on endothelium of all large venules was observed in tissues of LPS and TNF-alpha treated animals and staining for this newly synthesized P-selectin was enriched at the luminal surface of these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytogenetic analysis in ataxia telangiectasia with malignant lymphoma.

We present the results of cytogenetic analysis in a brother and sister with ataxia telangiectasia (AT), one of whom had malignant T-cell lymphoma. In both children, cytogenetic analysis of phytohemagglutinin (PHA)-stimulated lymphocytes showed chromosomal instability and inv(7) in 10% of the cells examined. The malignant lymphoma was analyzed cytogenetically on slides obtained from short-term culture of the lymph node cells; 64 cells were analyzed. A heterogeneous cell population was noted. Fourteen cells (21.9%) had a normal male karyotype; t(7;14)(p14;q12) and inv(7)(p14q35) were observed in 6.3% and 3.1% of metaphases. Owing to low frequency, these cells are probably a characteristic of the basic disease and have no features of malignant cells. Forty cells (62.5%) had a pseudodiploid karyotype 46,XY,dup(1)(p22p36),del(5)(q33),del(12)(p11), without cytogenetically evident aberrations of chromosomes 7 and 14. The results of these investigations suggest that the cells with rearrangements of chromosomes 1, 5, and 12 are malignant cells and did not originate by transformation of cells with inv(7) and t(7;14).