Umbilical cord blood CD34(+) stem cells and other mononuclear cell subtypes processed up to 96 h from collection and stored at room temperature maintain a satisfactory functionality for cell therapy.

BACKGROUND AND OBJECTIVES: Umbilical cord blood (UCB) is a good stem cell source for cell therapy. We recently demonstrated that cord blood mononuclear cell (MNCs) subtypes were viable and functional until 96 h after collection, even stored at room temperature. Now, we analyzed the viability and functionality of the cells before and after cryopreservation. MATERIALS AND METHODS: Twenty UCB units were analyzed at 24 and 96 h after collection, frozen for 6 months, thawed and re-evaluated. MNCs were analyzed by flow cytometry, viability by 7-AAD and clonogenic assays (CFU) were performed. RESULTS: After 96 h of storage, no substantial loss of MNC was found (median 7.320 × 10(6 ) × 6.05 × 10(6) ). Percentage and viability CD34(+) cells, B-cell precursors and mesenchymal stem cells were not affected. However, mature B and T lymphocytes as well as granulocytes had a substantial loss. CFU growth was observed in all samples. Prefreezing storage of 96 h was associated with a relative loss of colony formation (median 12%). Postthaw, this loss had a median of 49% (24 h samples) to 56% (96 h samples). CONCLUSION: The delay of 96 h before UCB processing is possible, without a prohibitive impairment of CD34(+) loss in number and functionality.

The impact of several phenotypic features at diagnosis on survival of patients with myelodysplastic syndromes.

Multiparametric flow cytometry is a useful co-criterion for diagnostic confirmation of MDS in patients with peripheral cytopenias and a normal karyotype. We examined the impact on patients' survival of several phenotypic aberrancies detected by a small 4-color panel of monoclonal antibodies (MoAbs). Diagnosis of the patients (54) was made by WHO criteria using peripheral blood counts, bone marrow (BM) morphology and karyotype. Flow cytometry was performed at diagnosis, and features obtained were compared to normal BM (24). We could detect 16 alterations: 4 in granulocytic precursors, 4 in monocytes, 6 in CD34+ cells, beside changes in plasmacytoid dendritic cells and basophil precursors. The total number of changes in RAEB was higher (median 8) than in cases with of abnormalities) were independent risk factors for a shorter survival. Our panel was sufficient to confirm the diagnosis of MDS and permitted to detect independent prognostic features.

A simple score derived from bone marrow immunophenotyping is important for prognostic evaluation in myelodysplastic syndromes.

Immunophenotyping of bone marrow (BM) precursors has been used as an ancillary diagnostic tool in myelodysplastic syndromes (MDS), but there is no general agreement about which variables are the most relevant for prognosis. We developed a parsimonious prognostic model based on BM cell populations well-defined by phenotype. We analyzed 95 consecutive patients with primary MDS diagnosed at our Institution between 2005 and 2012 where BM immunophenotyping had been performed at diagnosis. Median follow-up: 42 months (4-199). Median age: 67 years (33-79). According to IPSS-R, 71 cases were low or intermediate risk. Flow variables significant in the univariate Cox analysis: "%monocytes/TNCs", "% CD16+ monocytes/TNCs", "total alterations in monocytes", "% myeloid CD34+ cells", "number of abnormal expressions in myeloblasts" and "% of B-cell progenitors". In the multivariate model remained independent: "% myeloid CD34+ cells", B-cell progenitors" and "% CD16+ monocytes/TNCs". These variables were categorized by the extreme quartile risk ratio strategy in order to build the score: % myeloid CD34+ cells" (≥ 2.0% = 1 point), B-cell progenitors" (< 0.05% 1 point) and "CD16+ monocytes/TNCs" (≥ 1.0% 1 point). This score could separate patients with a different survival. There was a weak correlation between the score and IPSS-R. Both had independent prognostic values and so, the flow score adds value for the prognostic evaluation in MDS.

Variation of bone marrow CD34+ cell subsets in myelodysplastic syndromes according to who types.

Bone marrow (BM) hematopoietic progenitor cells (CD34+) are a heterogeneous population with varying degrees of commitment and maturation to several cell lineages. In myelodysplastic syndromes (MDS), this population is increased. We examined the major cell types found in the blast gate by flow cytometry in newly diagnosed patients with MDS, compared them to normal BM and studied their variation according to WHO type. Two subsets defined by SSC were found both in normal BM and MDS, corresponding to myeloblasts and B-cell precursors. The number of B-cell precursors among all nucleated cells was equally low, independent of WHO type. However, the subset with an intermediate SSC, but CD117, CD13 and CD19 negative increased with the rise of myeloblasts. Concomitantly, the ratio between CD34+/CD117+/CD34-/CD117+ cells was increased. These two features are consistent with the maturation block occurring in the progression of the neoplastic clone. We conclude that the quantitative analysis of the cell types present in the BM blast gate by flow cytometry is not only important for the diagnosis of MDS in patients with peripheral cytopenias and a normal karyotype, but gives also important prognostic information of the patients.

The prognostic value of maturation-associated phenotypic abnormalities in myelodysplastic syndromes.

Several phenotypic abnormalities of bone marrow (BM) hemopoietic precursors have been associated with disease progression in myelodysplastic syndromes (MDS). We analyzed the influence on overall survival of the expression of lineage and maturation-associated antigens of BM hemopoietic cells quantified in a previous study. In the univariate Cox regression the peripheral platelet count was a significant favourable factor for overall survival. Unfavorable prognostic factors were: WPSS, increase in BM CD34+ cells, increased mean fluorescence intensity (MFI) of CD13 on myelocytes, metamyelocytes and mature neutrophils as well as increased CD45 of myelocytes and mature neutrophils. In a model containing platelet count, WPSS and MFI of CD45 and CD13 on mature neutrophils, only hyperexpression of CD13 and degree of thrombocytopenia were independent risk factors. Therefore, phenotypic features that can also be obtained from PB might be useful for predicting survival in MDS.

Angiogenesis in salivary carcinomas with and without myoepithelial differentiation.

To investigate whether salivary carcinomas with and without myoepithelial differentiation could present differences regarding degree of angiogenesis, we compared tumor vascularization between adenoid cystic (31 cases) and epithelial-myoepithelial carcinomas (14) versus mucoepidermoid (37) carcinoma. The expression of peroxiredoxin I was also studied to verify the potential relationship between cellular metabolism and microvascular density. Microvascular density for CD34 and CD105 were significantly lower in carcinomas with myoepithelial differentiation. However, no correlation was found between degree of angiogenesis and amounts of myoepithelial cells. High-grade peroxiredoxin I expression was found in 73.7% of mucoepidermoid carcinomas, whereas 85.1% of carcinomas with myoepithelial differentiation presented low-grade expression. In conclusion, carcinomas with myoepithelial differentiation, regardless of the amounts of myoepithelial cells, are associated to a significantly lower vascular density. The reasons for this lower angiogenic activity remain to be determined but could be related to metabolic characteristics of the cancer cells.

Normal variation of bone marrow B-cell precursors according to age - reference ranges for studies in myelodysplastic syndromes in Brazil.

BACKGROUND: Normal B lymphoid maturation occurs in bone marrow (BM) throughout life, but immature B-cell progenitors (BCPs) are more numerous in children than in adults. To assess the normal values according to age became important as BCPs are decreased in myelodysplastic syndromes and have been considered an important diagnostic and prognostic feature in these clonal disorders. METHODS: in a multicenter retrospective study from the Brazilian Group of Flow Cytometry we analyzed the variation of BCPs in normal BM according to age and technical peculiarities of each laboratory. We analysed of 45 BM donors and 89 cases examined for elucidation of transitory reactive cytopenias presenting a normal BM immunophenotyping. BCPs were enumerated as CD19+ /CD34+ /CD45dim /CD10+ cells (panel 1) or CD19+ /CD34+ /CD45dim cells (panel 2) among the total nucleated non-erythroid cells and as percentage of CD34+ cells. RESULTS: we included 134 cases. Panel 1 was applied in 88 cases and panel 2 was used in 46. Age range: 10 months to 89 years. In a multiple regression, % BCPs/total nucleated cells was an exponential function of age. Age explained alone 49.4% of the variance, while 'panel used' explained 1.8% and 'laboratory' explained 0.7%. Age explained only 24.9% of the variance of BCPs/CD34+ cells. CONCLUSIONS: in normal individuals, BM B-cell precursors varied mainly according to age, but were also dependent on technical peculiarities of operators and equipments. Analysis by phenotype and as percentage of total nucleated cells was more accurate and less susceptible to variation than evaluating % BCPs/total CD34+ cells. © 2017 International Clinical Cytometry Society.

Angiogenic switch during tumor progression of carcinoma ex-pleomorphic adenoma.

We analyzed the tumor vascularization in carcinomas ex-pleomorphic adenoma (CXPA) to investigate the angiogenic switch during the malignant transformation of pleomorphic adenoma (PA) to carcinoma and during tumor progression. In eight cases of early CXPA (intracapsular and minimally invasive tumors), eight of advanced CXPA (widely invasive tumors), and ten of PA without malignant transformation, tumor vascularization was assessed in histological samples by measuring total microvascular area (TVA) and microvessel density (MVD) using CD34 and CD105 antibodies. MVD for CD105 increased significantly during tumor progression, whereas this was not the case for CD34 MVD. Comparing widely invasive CXPA with and without myoepithelial differentiation, CXPA with myoepithelial differentiation showed a significantly lower number of CD105 positive vessels but revealed higher TVA values. In these tumors, the neoplastic cells usually formed larger hypovascularized aggregates that were often surrounded by large-sized vessels. In conclusion, the antibody CD105 reveals an angiogenic switch during the progression from adenoma to carcinoma in salivary glands. The degree of angiogenesis and the total vascular area have distinctive patterns in CXPA with and without myoepithelial differentiation. Low angiogenesis associated with high TVA value is more characteristic of CXPA with myoepithelial differentiation.

Optimization of CD34+ collection for autologous transplantation using the evolution of peripheral blood cell counts after mobilization with chemotherapy and G-CSF.

BACKGROUND: Peripheral blood progenitor cells (PBPC) collection after high dose chemotherapy can be influenced by several factors. We searched for parameters that may predict the best day to start harvesting of PBPC in order to collect most CD34+ cells with the least number of aphereses. METHODS: We studied patients who underwent mobilization chemotherapy for autologous transplantation. The influence of age, sex, diagnosis, number of previous chemotherapy cycles, peripheral blood (PB) counts at day of mobilization (D0), day of neutrophils <1.0 x 10(9) l(-1) and day of nadir and interval between both (delta) on harvesting was investigated. Multivariate linear correlation models were built to predict the best harvesting with principles of parsimony. In patients where sequential CD34+ cell count was performed, the theoretical day of peak was calculated by interpolation in polynomial regression. RESULTS: One hundred and thirty four patients entered the analysis: 36 Hodgkin's lymphoma (HL), 65 B-large cell lymphoma (NHL) and 33 multiple myeloma (MM). Day of harvesting correlated with nr CHT, hemoglobin on D0, day of granulocytes <1.0 x 10(9) l(-1), delta and dosis of mobilization therapy. The day of CD34+ peak could be calculated by the formula = (-0.41) x Hemoglobin D0 + (day peripheral CD34+ cells = 10 x 10(6) microl(-1)) x 0.99 + 7.8. This model could explain 81% of the variance of the peak day and was stable by bootstrap resampling. Day of peripheral CD34+ cells = 10 x 10(6) microl(-1) preceded the calculated peak by 3-9 days. CONCLUSIONS: Although the day of best collection can be predicted using only sequential PB counts after mobilization chemotherapy, a model of prediction using peripheral CD34+ cell count is important especially for optimizing collection in poor mobilizing patients.

Phenotypic quantitative features of patients with acute myeloid leukemia.

The recent WHO classification for acute myeloid leukemias (AML) separates entities by recurrent cytogenetic abnormalities and immunophenotypic features presenting prognostic impact. We have examined the expression of several lineage and maturation linked antigens used in routine immunophenotyping of patients with de novo AML, using a 3-color two-step panel. Cases were diagnosed by peripheral blood counts, bone marrow cytology, cytochemistry, cytogenetics and immunophenotyping (CD2, CD3, CD7, CD19, CD13, CD33, myeloperoxydase -- MPO, CD14, CD15, HLA-DR, CD34, CD56 and CD45). Antigen expression was measured by mean fluorescence intensity (MFI) by flow cytometry (Paint-a-gate software). Thirty five patients were analyzed. Median age: 51 years (15-79). Predominant FAB types were M2 and M4. In 6 cases more than one phenotypically distinct blast subpopulation could be detected. Although our set was small, we tried to analyze the impact of MFI of the examined antigens on the overall survival of the patients. In Cox univariate analysis, age, peripheral leukocytes (WBC) at diagnosis, MFI of CD45, and MPO were significant for worse a survival. In the multivariate analysis only MFI of CD45 and WBC remained in the model (p=0.018 and p=0.014 respectively). After bootstrap resampling, MFI of CD45 entered the model in 69%, WBCin 60%, age in 42% and MFI of MPO in 35% of the sets. Analysis of antigen expression by MFI permitted to detect cases presenting phenotypically distinct blast subpopulations. This may represent a pitfall in studies of minimal residual disease by flow cytometry, as chemotherapy may select one of these subsets.

Effects of vitamin E on endothelium-dependent coronary flow in hypercholesterolemic dogs.

The authors studied the effect of vitamin E on endothelium-dependent coronary flow in hypercholesterolemic dogs. Adult mongrel dogs weighing 7.4 +/- 1.0 kg were divided into control, hypercholesterolemic and vitamin E groups. The animals in the hypercholesterolemic group were fed a diet enriched with cholesterol (5% w/w) and coconut oil (10% w/w) for 40 days. The vitamin E group received the same diet plus 400 IU of vitamin E during the last 15 days of the experiment. Total serum cholesterol levels were evaluated at the beginning and at the end of the experiment using a commercial enzyme kit and a Beckman analyzer. The coronary flow was determined by electromagnetic flowmetry using a probe positioned in the left anterior descending coronary artery, near the ostium. A needle connected to a perfusion pump was introduced into the coronary artery for the administration of acetylcholine and sodium nitroprusside at a rate of 5 micrograms/kg per min. The aorta was cannulated for the measurement of arterial blood pressure via a pressure transducer coupled to a Siemens multi-channel recorder. The tissue cholesterol content and malonic dialdehyde (MDA) were also measured in isolated coronary vessel specimens. At the end of 40 days, the serum cholesterol levels had increased by 226% and 190% in the hypercholesterolemic and vitamin E groups, respectively. However, the difference in the levels of these two groups was not significant (P > 0.05). The aortic blood pressure and heart rate remained unchanged during acetylcholine administration. In contrast, systolic and diastolic pressure fell and the heart rate increased during the infusion of sodium nitroprusside. The tissue cholesterol content and MDA were significantly (P < 0.05) increased in coronary artery specimens from the hypercholesterolemic compared to control animals. Vitamin E was able to reduce these increases in cholesterol treated animals (P < 0.05). The percent change in coronary flow during acetylcholine administration was significantly lower in the hypercholesterolemic group when compared with control animals (P < 0.05) but was unaltered in the vitamin E group (P > 0.05). During sodium nitroprusside administration, the coronary flow increased in the vitamin E group (P < 0.05). The authors conclude that hypercholesterolemia reduces endothelium-dependent coronary flow and increases the tissue cholesterol content and MDA of coronary arteries. Vitamin E decreases the MDA and the tissue cholesterol content without significantly affecting the total serum cholesterol level. Vitamin E may thus restore coronary flow by reverting endothelial dysfunction.

Attitudes of medical students to necropsy.

AIM: To compare the attitudes of students towards the necropsy at different stages of their undergraduate career. METHOD: Students in the first, fourth and sixth academic years (n = 283) were asked to respond anonymously to a questionnaire comprised of 26 attitude statements. These statements dealt with the importance of the necropsy in medicine, rapport with the bereaved family and emotional reactions to the necropsy. RESULTS: Of the students, 226 (80%) completed the questionnaire. Overall, the students agreed on/the importance of the necropsy. The three groups differed in 10 statements on the approach to the bereaved family and emotional reactions to the necropsy. First year students showed more personal involvement and would have more difficulties in approaching the family of the deceased as well as in attending a necropsy. These reactions were increasingly less noticeable with fourth and sixth year students. The latter group was also more inclined to accept cremation, organ donation and necropsy of their own corpses. CONCLUSION: The changes in attitudes towards the necropsy throughout undergraduate study may reflect both the influence of psychological defense mechanisms and the viewing of necropsy as a relevant tool in medical practice. Necropsy should be carefully and sensitively incorporated into programmes designed to teach students about death and dying. This might reduce both their reluctance to seek permission for necropsy and their difficulty in looking after the dying patient.

Pathology of patients with Chagas' disease and acquired immunodeficiency syndrome.

The main pathologic findings in 23 patients with acquired immunodeficiency syndrome (AIDS) and Chagas' disease are reviewed; five are from our own experience and 18 from the literature. The presence of Trypanosoma cruzi parasites and/or T. cruzi antibodies in blood and cerebrospinal fluid was recorded and computerized tomograms of the brain were evaluated. Twenty (87%) of the 23 subjects developed severe, multifocal or diffuse meningoencephalitis with necrosis and hemorrhage associated with numerous tissue parasites. The second most severely affected site was the heart. Seven (30.4%) of the 23 cases had myocarditis on pathologic examination. It was acute in four patients, chronic in two, and simultaneously acute and chronic in one. Acute myocarditis and meningoencephalitis are interpreted as being caused by relapses of chronic T. cruzi infections. An AIDS permissive role is suggested for these conditions since immunologic defense against T. cruzi is mediated mainly by T lymphocytes, whose CD4 subpopulation is depleted in patients with this disease. Consequently, AIDS is a factor that may favor the reactivation of T. cruzi infections. The lesions reported in the association of Chagas' disease with AIDS were compared with those reported from patients without AIDS having fatal, acute, vector-transmitted infections, contaminated blood transfusions, or accidental exposures in the laboratory. For the latter three, meningoencephalitis is uncommon. Only immunosuppressed cases of Chagas' disease have been described as having a pseudotumoral presentation that shows expanding lesions with a mass effect in the cranial cavity that causes intracranial hypertension and simulates neoplasms (tumors such as gliomas, lymphomas, metastases, etc.).

Enalapril does not prevent the myocardial ischemia caused by the chronic inhibition of nitric oxide synthesis.

In rats, chronic administration of the nitric oxide (NO) inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) causes arterial hypertension, cardiac hypertrophy and myocardial ischemic alterations such as necrosis and fibrosis. In this study, we evaluated the effect of 8 weeks of treatment with enalapril maleate on cardiac weight and on the development of the histological alterations induced by L-NAME. Enalapril significantly inhibited the development of both arterial hypertension (117.2 +/- 5.8, 161.8 +/- 8.8 and 122.0 +/- 10.6 mm Hg, for control, L-NAME- and L-NAME + enalapril-treated animals, respectively) and left ventricular hypertrophy (1.36 +/- 0.13, 1.60 +/- 0.04 and 1.48 +/- 0.05 mg/g, for control, L-NAME- and L-NAME + enalapril-treated animals, respectively), but had no effect on the myocardial lesions. These findings demonstrate that although the renin-angiotensin system plays a major role in the development of arterial hypertension and cardiac hypertrophy, it does not modulate the ischemia-induced myocardial alterations observed in this model.

Effect of Ca2+ channel blockers on arterial hypertension and heart ischaemic lesions induced by chronic blockade of nitric oxide in the rat.

The effects of the Ca2+ channel blockers diltiazem, nifedipine and amlodipine were investigated on both arterial hypertension and myocardial changes induced by chronic blockade of nitric oxide synthesis. Control male Wistar rats received Nomega-nitro-L-arginine methyl ester (L-NAME; 20 mg rat(-1) day(-1)) in the drinking water for 8 weeks; blood pressure and body weight were monitored weekly. The Ca2+ channel blockers were given concomitantly to L-NAME, as follows: diltiazem (13.5 mg rat(-1) day(-1)) and amlodipine (6.25 mg rat(-1) day(-1)) were administered in the drinking water whereas nifedipine (6.25 mg rat(-1) day(-1)) was given in the chow. Nomega-nitro-L-arginine methyl ester induced a time-dependent increase in blood pressure which was significantly attenuated by diltiazem (154+/-1.6 vs. 139+/-1.6 mm Hg, p < 0.05), nifedipine (166+/-2.7 vs. 150+/-2.1 mm Hg, p < 0.05) and amlodipine (208+/-5.8 vs. 158+/-1.8 mm Hg, p < 0.05) at the last week of the treatment. Rats treated with the L-NAME also developed myocardial ischaemia, as indicated by the increased percentage of fibrous tissue found in the left ventricles of these animals (10.9+/-0.1%, p < 0.01) when compared to control ones (6.3+/-0.1%). Neither diltiazem (14.9+/-1.2%) nor nifedipine (11.1+/-1.5%) prevented this effect whereas amlodipine (6.9+/-1.1%, p < 0.01) virtually abolished the increase in fibrous tissue induced by L-NAME. The plasma concentration of the Ca2+ channel blockers was measured by liquid chromatography coupled to mass spectrometry at two different time points (morning and afternoon). Only amlodipine treatment was able to maintain constant levels (186+/-46 ng ml(-1) in the morning and 110+/-19 ng ml(-1) in the evening) compared to nifedipine (3003+/-578 ng ml(-1) in the morning and 436+/-100 ng ml(-1) in the evening) and diltiazem (77+/-51 ng ml(-1) in the morning and not detectable in the evening). In conclusion, our results indicate that amlodipine (but not diltiazem and nifedipine) can efficiently control myocardial ischaemia in nitric oxide deficient rats, probably due to its intrinsically long half-life.

Comparison of immunoexpression of 2 antibodies for estrogen receptors (1D5 and 6F11) in breast carcinomas using different antigen retrieval and detection methods.

The importance of in situ immunodetection of hormone receptors for therapy planning and prognostic evaluation in patients with breast carcinoma is well established. Sensitive detection methods are of utmost importance, especially in poorly fixed tissues, which are not uncommon in routine pathologic practice. The purpose of the present study is to compare immunoexpression of estrogen receptors in 20 cases of invasive ductal carcinoma using two antibodies, 1D5 and 6F11, and to verify the effect of different antigen retrieval solutions and detection systems. Immunoperoxidase was performed on paraffin sections using 1D5 and 6F11 as primary antibodies. Heat-induced antigen retrieval was performed using citrate buffer (pH 6.0) or Tris-EDTA buffer (pH 8.9). Detection was achieved using the following systems: EnVision, EnVision Plus, and labeled streptavidin-biotin peroxidase complex. Reaction was semiquantified from 0 to 4. There were no differences between the two markers, 1D5 and 6F11, except when 6F11 was used with EnVision and citrate buffer, in which case weaker reactivity was observed. Only in this combination (6F11/EnVision) was EDTA buffer significantly better than citrate. Labeled streptavidin-biotin peroxidase complex presented the best results, followed by EnVision Plus.

Atypical stromal giant cells of cervix uteri--evidence of Schwann cell origin.

The report describes atypical multinucleated giant cells adjacent to proliferated nerve fascicles in a circumscribed subepithelial area of the cervix uteri of a 44-year-old multipara. Ultrastructural examination revealed cytoplasmatic processes, basal lamina, intracytoplasmic microfibrils, bizarre nuclear shapes with pseudoinclusions and nuclear fragments connected by small chromatin bridges (nucleotesimals). Immunohistochemical examination showed positive staining for vimentin and S-100 protein. Quantitative topography exhibited an isotropic distribution of the giant cells in an anisotropic architecture of mononuclear cells. A Schwann cell origin of the atypical giant cells is postulated. Aetiopathogenetically the lesion is regarded to be due to a trauma during delivery followed by regenerative proliferation of nerve fascicles and degenerative alterations of proliferating Schwann cells. The knowledge of this lesion is considered important, because the atypical cells could be confounded with malignant neoplastic cells.

Microphotometric quantitation of enzyme activities in giant cell tumor of bone. A case study.

In a proliferating giant cell tumor of bone the activities of tartrate-resistant acid phosphatase (acPase) and of NADH-tetrazolium reductase were demonstrated by enzyme histochemical methods. Quantitative microphotometry made it possible to determine the relative enzyme activities per given volume unit in the cytoplasm of giant cells of several sizes. The activity of tartrate-resistant acid phosphatase increases with increasing cell size, whereas the activity of tetrazolium reductase will decrease in proportion. This coincidence of high acPase activity and low tetrazolium reductase activity in larger giant cells is interpreted as an expression of degenerative change.

Size dependent enzyme activities of multinucleated (osteoclastic) giant cells in bone tumors.

In osteoclastic giant cells of six different tumors of bones and joints (fibrous dysplasia, proliferating giant cell tumor, malignant giant cell tumor, osteosarcoma after chemotherapy, malignant synovioma and Ewing's sarcoma) activities of tartrate-resistant acid phosphatase, NADH-tetrazolium-oxidoreductase and, in three of them, of non-specific esterase are determined by enzyme histochemical methods. Quantitative microphotometry makes it possible to determine relative enzyme activities in the cut sections of giant cells of different sizes. Giant cells of the various tumors reveal similar trends: With an increase in cell size, mean extinctions of NADH-tetrazolium-oxidoreductase and non-specific esterase decrease. Mean extinctions of tartrate-resistant acid phosphatase increase in cells of medium size, whereas the large cells reveal in part low activities. An additional ultrastructural examination of the giant cells in the proliferating giant cell tumor as well as in the osteosarcoma shows morphological signs of degeneration in the large cells. Electron probe microanalysis of the proliferating giant cell tumor exhibits evidence of phagocytosis of Ca and/or Fe containing particles. The similar size dependent reaction pattern of enzymes in osteoclastic giant cells of different tumors favors the concept of a common histogenesis, i.e. a host reaction.

The value of PCNA and AgNOR staining in endoscopic biopsies of gastric mucosa.

The aim of the present study was to examine the usefulness of the quantification of PC10-positive-cells and of Argyrophilic Nucleolar Organizer Regions (AgNORs) in gastric biopsies for the identification of gastric mucosal proliferative lesions. Fifty seven paraffin-embedded endoscopic biopsies were classified into four histologic groups: normal, inflammatory, dysplastic and neoplastic mucosa. The percentage of PC10-positive cells was determined by immunohistochemistry. The AgNOR parameters determined included the total number of all identifiable silver precipitations in the nucleus, the mean number of silver precipitations per cluster, and the presence of morphologically heterogenous silver precipitations. Group comparisons were performed using the Kruskall Wallis and Dunn non-parametric tests with a significance level of 5%. A discriminant analysis (followed by the jack-knife procedure) was performed using the three AgNOR parameters plus the percentage of PCNA-positive cells as the independent variables and histological groups as the dependent variable. All three AgNOR parameters, as well as the percentage of PCNA-stained nuclei, showed their highest values in the carcinoma group. However, no good differentiation among the four histologic groups was obtained using only one of these parameters, since there was always considerable overlap among them. By combining all the parameters in a linear discriminant analysis, we obtained a correct classification in 48 out of 57 cases. Within the classification errors there was only one false positive carcinoma, which was in fact a dysplasia and only one false negative carcinoma erroneously classified as dysplasia. The number of cells with heterogenous AgNORs was the most important parameter for the discriminant analysis. No correlation between PCNA values and the AgNOR parameters could be found, thus indicating that they do not represent the same phenomenon in the cell cycle. We concluded that the use of a combination of various proliferation parameters in a linear discriminant analysis may be helpful for differentiating gastric mucosal lesions. The peculiar AgNOR morphology is an important variable which should be taken in consideration in quantitative studies. PCNA and AgNORs seem to represent different physiological phenomena in the cell cycle.