Digital cytology: A short review of technical and methodological approaches and applications.

The recent years have been characterised by a rapid development of whole slide imaging (WSI) especially in its applications to histology. The application of WSI technology to cytology is less common because of technological problems related to the three-dimensional nature of cytology preparations (which requires capturing of z-stack information, with an increase in file size and usability issues in viewing cytological preparations). The aim of this study is to provide a review of the literature on the use of digital cytology and provide an overview of cytological applications of WSI in current practice as well as identifying areas for future development.

Quantitative assessment of 99mTc-depreotide uptake in patients with non-small-cell lung cancer: immunohistochemical correlations.

BACKGROUND: Somatostatin receptor (SSTR) scintigraphy with (99m)Tc-depreotide is used for differential diagnosis of solitary pulmonary nodules. The method is based on SSTR expression in cancer tissue. PURPOSE: To estimate the expression of SSTRs in non-small-cell lung cancer (NSCLC) in vitro, and to determine the correlation between (99m)Tc-depreotide uptake in vivo and different tumor characteristics determined in vitro, such as tumor grade, and presence of SSTR2, MIB-1, and p53. MATERIAL AND METHODS: A total of 127 patients with lung lesions detected on computed tomography (CT) were investigated with SSTR scintigraphy after injection of 740 MBq (99m)Tc-depreotide. This study includes 19 patients with NSCLC with histologically proven diagnosis. The quantitative evaluation of (99m)Tc-depreotide was performed using region-of-interest analysis and includes tumor counts/cm(3), background counts/cm(3), and the ratio between tumor and background counts. RESULTS: 99mTc-depreotide uptake was found in all NSCLC tumors, which expressed SSTR2 defined in vitro by immunochemical methods. SSTR2 expression was negatively correlated to the degree of the tumor's differentiation (P<0.05). 99mTc-depreotide uptake in tumor cells did not correlate with tumor grade, or SSTR2, MIB-1, or p53 expression. CONCLUSION: There is an expression of SSTRs in NSCLC. The degree of tumor differentiation correlates negatively with SSTR2 measured in vitro and positively with MIB-1 expression in tumor tissue. No correlation was found between (99m)Tc-depreotide uptake and possible prognostic factors such as MIB-1 and p53 expression in tumor cells in NSCLC. Lastly, no correlation was found between (99m)Tc-depreotide uptake and tumor grade or SSTR2 expression.

Ossifying fibromyxoid tumor with atypical histological features: a case report.

Ossifying fibromyxoid tumor of soft tissues (OFMT) is considered a rare mesenchymal neoplasm. Its main histological features are sheets and ill-defined lobules of rounded bland cells within a fibromyxoid background and a thick collagenous capsule with an incomplete rim of lamellar bone. This lesion occurs mostly in the soft tissues of the lower extremities and limb girdles. In this paper, we describe a mesenchymal tumor removed from the right thigh of a 41 year-old-woman. The neoplasm differed histologically from typical forms of OFMT for areas of moderate cellularity and atypia, nuclear enlargement and small nucleoli. Focally, stromal tongues of osteoid were centrally and irregularly located within the lesion with evident spindling of tumor cells around them. The mitotic activity was low (up to 19 per 50 HPF) and atypical figures were rarely seen. The tumor was positive to S-100 protein, vimentin, CD10, CD56, CD99, ASMA, calponin and collagen IV. Rare elements were positive for cytokeratin AE1/AE3. To the best of our knowledge, this is the first case of atypical OFMT reported to be positive for calponin. The patient is currently alive and well with no evidence of disease at 96 months following surgery. In spite of low-grade histology, OFMT has high local recurrence rate and low metastatic potential, primarily in the lungs, even several years after surgical removal. The recognition of this entity is important. In this report the authors address differential diagnosis and enigmatic histogenesis of this neoplasm.

Increased expression of specific thioredoxin family proteins; a pilot immunohistochemical study on human hepatocellular carcinoma.

Hepatocellular Carcinoma (HCC) is one of the most frequent cancers worldwide, however, prognosis remains poor following its discovery. We investigate the Thioredoxin superfamily of proteins as diagnostic markers for HCC. Furthermore, we delineate possible roles of the endoplasmic reticulum member of the superfamily, ERdj5, in carcinogenesis. Using antibodies against Thioredoxin 1, Thioredoxin Reductase 1 and ERdj5, we performed immunohistochemistry on paraffin embedded liver biopsy sections from HCC patients. All three redox proteins exhibited elevated expression levels in tumor tissue compared to internal control, with ERdj5 showing a remarkable 3-fold increase. In vitro cell viability experiments using Hepatocellular Carcinoma HuH7 cells treated with ERdj5 small interfering RNA showed that ERdj5 knockdown cells exhibited less resistance to Doxorubicin (chemotherapy drug), but more resistance to Tunicamycin (Endoplasmic Stress inducer), compared to control cells. In conclusion, we introduce members of the Thioredoxin superfamily as possible immunohistochemical markers in the diagnostics of hepatocellular carcinoma and indicate a potential defensive role for ERdj5 in chemotherapeutic drug resistance.

[Lymphoepithelioma-like carcinoma of the esophagus: description of a case]. / Carcinoma simil linfoepitelioma (LELC) dell'esofago: descrizione di un caso.

The authors describe a case of lymphoepithelioma-like carcinoma (LELC) of the esophagus occurring in a 77-year-old man. To date, 13 cases have been described in the literature, all in natives of Japan. Histological features, immunohistochemical findings and differential diagnosis are discussed. In our case epithelial neoplastic cells were completely negative for the EBV genome.

[Lympho-epithelial carcinoma (malignant lympho-epithelial lesions) of the parotid gland. Description of a case with detection of EBV by in situ hybridization]. / Carcinoma linfoepiteliale (lesione linfoepiteliale maligna) della ghiandola parotide. Descrizione di un caso con rilevazione dell'EBV mediante ibridazione in situ.

The authors describe a case of malignant lymphoepithelial lesion (MLEL), commonly referred to as lymphoepithelial carcinoma of parotid gland, that is a very rare tumour. There is a relatively high incidence in Eskimos of Alaska and Greenland, but some cases are described in natives of south China. The immunophenotypic profile and histopathological aspect of this neoplasm are discussed, and the differential diagnosis in regard to other primitive or metastatic tumours of parotid is also considered. In our case a diffuse positivity of epithelial neoplastic cells for EBV genome was found using in situ hybridization. The possible role of EBV in the ethiopathogenesis of this rare lesion is herein discussed.

A morphometric analysis of cytological features of tall cell variant and classical papillary carcinoma of the thyroid.

In order to assess whether morphometric parameters could be of value in distinguishing between tall cell variant and classical pattern of thyroid papillary carcinoma, the fine needle aspiration cytology (FNAC) samples of 14 cases were analysed using Arcimage 5 software on an Acorn computer. Histological examination of the specimens allowe classification of nine of them as classical pattern and the remaining five as tall cell variants. The nuclear diameter (NDD) and standard deviation distribution (NDSDD), th nuclear area (NAD) and standard deviation distribution (NASDD), and the nuclear/cytoplasmic ratio (NCR) were assessed on May-Grunwald-Giemsa stained smears. Statistical analysis was performed by use of one-way analysis of variance (ANOVA) of the two groups as identified by histology. Whilst NDD (P = 0.007), NAD (P = 0.015) and NADSD (P = 0.026) all appeared statistically significant, NDSD (P = 0.06) and NCR (P = 0.71) were not. The cytological diagnosis of papillary carcinoma is established and reproducible, but morphometric data on the thyroid have so far focused on the differential diagnosis between benign and malignant nodules. The choice of simple morphometric parameters appears to be helpful in the preoperative distinction between the classical pattern and tall cell variant of papillary carcinoma.

[Microcalcifications associated with in situ ductal breast carcinoma. Mammographic and histological correlations with morphometric evaluation]. / Microcalcificazioni associate a carcinoma mammario duttale in situ. Correlazioni mammografiche e istologiche con valutazioni morfometriche.

This study includes 33 cases of ductal carcinoma in situ and its aim is to detect and classify microcalcifications according to their appearance, mammographically, histologically and morphometrically. From the histological point of view, intraductal carcinomas are classified following the criteria proposed by Holland et al. The types of carcinoma examined reveal the presence of different microcalcifications. The calcifications associated with G1 carcinomas appear as highly compact morphometrically, fine or mainly so mammographically and laminated or mixed histologically; no granular calcifications are observed. On the other hand, calcifications associated with type G2 appear as coarse mammographically, granular histologically and scarcely compact morphometrically. Finally, G3 carcinomas mainly reveal by mammography vermicular calcifications (in a few cases associated with fine ones). Granular calcifications are predominant on histological examination while morphometry shows the poorest level of compactness.

Preoperative diagnostic accuracy of fine-needle aspiration in the management of breast lesions: comparison of specificity and sensitivity with clinical examination, mammography, echography, and thermography in 249 patients.

Two hundred and forty-nine women suffering from breast problems underwent a complete series of tests including clinical examination, mammography, echography, thermography, and fine-needle aspiration (FNA). Ninety-four of these patients were shown to be positive or to have suspected malignancy. Accordingly, they underwent surgical excision followed by histologic examination, while the remaining patients were re-examined after 12 to 18 mo in order to exclude false negatives. The analysis of specificity and sensitivity of every single procedure showed that FNA describes the best degree of sensitivity and specificity but no procedure allows, by itself, the detection of all carcinomas. When considered in combination, clinical examination, mammography, and fine-needle aspiration have a sensitivity of 100% and a specificity of 49%, and are the best diagnostic tests for a correct assessment of mammary lesions. Thermography and echography showed a low degree of sensitivity and should not be included in the routine diagnostic procedure of breast lesions.

Ticlopidine selectively inhibits human platelet responses to adenosine diphosphate.

Platelet aggregation and fibrinogen binding were studied in 15 individuals before and 7 days after the oral administration of ticlopidine (250 mg b.i.d.). Ticlopidine significantly inhibited platelet aggregation induced by adenosine diphosphate (ADP), the endoperoxide analogue U46619, collagen or low concentrations of thrombin, but did not inhibit platelet aggregation induced by epinephrine or high concentrations of thrombin. Ticlopidine inhibited 125I-fibrinogen binding induced by ADP, U46619 or thrombin (1 U/ml). The ADP scavengers apyrase or CP/CPK, added in vitro to platelet suspensions obtained before ticlopidine, caused the same pattern of aggregation and 125I-fibrinogen binding inhibition as did ticlopidine. Ticlopidine did not inhibit further platelet aggregation and 125I-fibrinogen binding induced in the presence of ADP scavengers. After ticlopidine administration, thrombin or U46619, but not ADP, increased the binding rate of the anti-GPII b/III a monoclonal antibody 7E3 to platelets. Ticlopidine inhibited clot retraction induced by reptilase plus ADP, but not that induced by thrombin or by reptilase plus epinephrine, and prevented the inhibitory effect of ADP, but not that of epinephrine, on the PGE1-induced increase in platelet cyclic AMP. The number of high- and low-affinity binding sites for 3H-ADP on formalin-fixed platelets and their Kd were not modified by ticlopidine. These findings indicate that ticlopidine selectively inhibits platelet responses to ADP.

[Suprapubic bladder ultrasonography and urinary cytology: indications and limits in the follow-up of superficial bladder tumors]. / Ultrasonografia vescicale sovrapubica e citologia urinaria: indicazioni e limiti nel follow-up dei tumori superficiali della vescica.

139 patients underwent urinary cytology and bladder sonography in follow-up of superficial bladder cancer (Ta G1-3) alternatively or at the same time of cystoscopy. Medium follow-up was 27.2 mos. In 7.91% there was progression to T1 o T2 but no case escaped this protocol. In 9% urinary cytology and bladder sonography were both falsely negative: tumors were smaller than 0.5 cm and low grade. In 76 patients with Tar bladder cystoscopy rate was 1/5.2 mos. before this study and 1/7.2 mos. after this study. In our opinion this protocol reveals the recurrence of superficial bladder tumor, reduce cystoscopy rate with no risk of ignored progression.

Pseudopathologies of hemostasis and dental surgery.

Values of hemostasis tests are grossly abnormal in persons with factor XII deficiency, dysfibrinogenemia, the lupuslike anticoagulant, and pseudothrombocytopenia, yet these persons do not usually bleed spontaneously or even after surgery. We reviewed the records of 66 patients with laboratory diagnosis of one of the aforementioned abnormalities to determine whether they bled excessively after challenges to hemostasis such as dental extractions. Only three of them bled excessively, but two of these had other concomitant causes that could explain bleeding. Hence the dentist should not give up dental extractions or other surgical procedures when results of hemostasis tests are grossly abnormal, because these are not necessarily associated with abnormal hemostasis. Obviously, the correct interpretation of abnormal hemostasis values warrants close collaboration and reciprocal consultation between the dentist and the hematologist.

[Pseudopathology of hemostasis in dental practice]. / Pseudo patologie dell'emostasi nella pratica odontostomatologica.

The authors demonstrate, with a 66 patients research, that also when laboratory tests on hemostasis indicate pathology like XII factor penia, disfibrinogenemy, lupus (Lac), pyastrinopenia, we manage pseudopathology. In these cases haemostasis is completely normal, even after surgery, like tooth extraction.

Fibrinogen interaction with platelets of diabetic subjects.

Fibrinogen-platelet interaction was studied in suspensions of platelets obtained from patients with uncontrolled diabetes mellitus of long duration and from control individuals. Fibrinogen binding sites were exposed by stimulating platelets with ADP or with chymotrypsin. There was no significant difference in fibrinogen mediated aggregation between ADP-stimulated platelets of 80 control and 47 diabetic subjects. The Km values for fibrinogen mediated aggregation of ADP-stimulated platelets obtained from control and diabetic donors were 1.39 +/- 0.13 X 10(-7)M and 1.44 +/- 0.13 X 10(-7)M; the Vmax values (expressed in arbitrary light transmission units) were 87.8 +/- 3.14 and 92.8 +/- 4.5 (mean +/- S.E.M.). The analysis of variance showed no significant relationship between Km, Vmax, age and sex in control group; in patient group there was no significant relationship between Km, Vmax, age, sex, type of diabetes, presence of vascular complications and type of treatment (insulin and/or oral hypoglycemic agents). Fibrinogen mediated aggregation of chymotrypsin-treated platelets showed similar pattern in 25 control and in 25 diabetic donors. In 24 normal individuals and in 24 diabetic patients Scatchard analysis revealed 48,820 +/- 5350 fibrinogen binding sites per one normal platelet (Kd = 4.7 X 10(-7)M) and 45,350 +/- 4663 sites per one diabetic platelet (Kd = 3.5 X 10(-7)M). Our data suggest a normal pattern of interaction between fibrinogen and fully activated platelets of diabetic subjects.

Binding of fibrinogen to human monocytes.

The interaction of fibrinogen with monocytes was studied. After stimulation with ADP (10 microM) or thrombin (1 U/ml), platelet-free suspensions of human monocytes bind 125I-fibrinogen with two different affinities in a specific and Ca2+-dependent reaction with saturation at 5.80-7.35 X 10(-7) M of added protein. The binding of fibrinogen to specific receptors on monocytes induces the procoagulant activity of these cells. Thrombasthenic cells or normal monocytes preincubated with a monoclonal antibody to the platelet glycoprotein IIb/IIIa complex (10E5) do not bind fibrinogen and have no procoagulant activity. Metabolic studies with [35S]methionine revealed that cultured monocytes actually synthesize a surface antigen precipitated by 10E5 antibody as a major band with 92,000 relative molecular weight. Our data indicate that monocytes express receptors for fibrinogen only in part related to the platelet glycoprotein IIb/IIIa complex. Furthermore, the binding of fibrinogen to monocytes enhances the cooperation of these cells in hemostasis.

von Willebrand factor contaminating porcine factor VIII concentrate (Hyate:C) causes platelet aggregation.

Severe thrombocytopenia has been reported to occur in some patients treated with a polyelectrolyte-fractionated porcine factor VIII concentrate (Hyate:C) (Kernoff et al, 1984; Gatti & Mannucci, 1984). We report here that Hyate:C induces aggregation of platelet-rich plasma in vitro accompanied by ATP release and thromboxane B2 formation, and is inhibited by EDTA and prostaglandin E1. Hyate:C induced platelet aggregation is affected by monoclonal antibodies anti-glycoprotein Ib or anti-glycoprotein IIb/IIIa and suppressed by polyclonal anti-porcine von Willebrand factor antiserum. We conclude that the aggregating ability of Hyate:C is due to porcine von Willebrand factor present in the concentrate. The occasional thrombocytopenia described after Hyate:C infusions probably results from the interaction between platelets and the porcine von Willebrand factor contaminating the concentrate.

A myeloma paraprotein with specificity for platelet glycoprotein IIIa in a patient with a fatal bleeding disorder.

Impaired platelet aggregation, normal shape change, and agglutination and normal ATP secretion and thromboxane synthesis in response to high concentrations of thrombin or arachidonic acid were found in a patient with multiple myeloma and hemorrhagic tendency. The purified IgG1 kappa or its F(ab1)2 fragments induced similar changes when added in vitro to platelet-rich plasma from normal subjects. In addition, the paraprotein inhibited adhesion to glass microbeads, fibrin clot retraction, and binding of radiolabeled fibrinogen or von Willebrand factor to platelets exposed to thrombin or arachidonic acid without affecting intraplatelet levels of cAMP. The radiolabeled para-protein bound to an average of 35,000 sites on normal platelets but it bound to less than 2,000 sites on the platelets from a patient with Glanzmann's thrombasthenia. Immunoprecipitation studies showed that the platelet antigen identified by the paraprotein was the glycoprotein IIIa. Furthermore, binding of radiolabeled prostaglandin E1 (PGE1) to resting platelets as well as binding of von Willebrand factor to platelets stimulated with ristocetin were entirely normal in the presence of patient's inhibitor. These studies indicate that bleeding occurring in dysproteinemia may be the result of a specific interaction of monoclonal paraproteins with platelets. In addition, our data support the concept that the interaction of fibrinogen and/or von Willebrand factor with the platelet glycoprotein IIb-IIIa complex is essential for effective hemostasis.

Interaction of platelet factor 4 with human platelets.

Human washed resting platelets bound 125I-labeled platelet factor 4 in a reaction which was saturable and approached equilibrium within 15-30 min. Scatchard plot analysis of the binding isotherms suggested a single class of specific binding sites. Excess of unlabeled protein and low- and high-affinity heparin competed for platelet factor 4 binding sites on the platelet surface and caused a partial displacement of this molecule. Anti-platelet factor 4 Fab fragments caused inhibition of binding of 125I-platelet factor 4 to platelets. Most of the labeled platelet factor 4 which was bound to intact platelets was recovered in the Triton X-100-insoluble cytoskeletal fraction prepared from the same platelets after their stimulation by thrombin. The association with the cytoskeleton was inhibited by anti-platelet factor 4 Fab fragments and by low-affinity heparin. Anti-platelet factor 4 125I-labeled Fab fragments bound to resting platelets, and this binding was greatly increased following platelet stimulation with thrombin. This suggested that endogenously secreted platelet factor 4 also binds to the platelet surface. No significant binding to platelets of 125I-labeled beta-thromboglobulin and 125I-labeled anti-beta-thromboglobulin Fab fragments was observed. Fab fragments of monospecific anti-human platelet factor 4 antibody raised in rabbits inhibited platelet aggregation and secretion induced by low concentrations of thrombin. Fab fragments of anti-beta-thromboglobulin antibody had no inhibitory effect. We suggest that the binding of alpha-granule-derived platelet factor 4 to the specific sites on the surface of platelets may modulate platelet aggregation and secretion induced by low levels of platelet agonists.

Exposure of fibrinogen receptors on fresh and stored platelets by ADP and epinephrine as single agents and as a pair.

Platelet concentrates stored at 22 degrees C have a marked decrease in their aggregation response to adenosine diphosphate (ADP) or epinephrine but a normal response to these agents when used as a pair. Since platelet stimulation involves exposure of receptors for fibrinogen, we studied fibrinogen binding to platelets from fresh and stored concentrates. Following stimulation with 10 microM ADP or 20 microM epinephrine, platelet suspensions from fresh concentrates bound 125I-fibrinogen in a reaction that reached completion within 30 min. Significantly less binding occurred in suspensions from platelet concentrates that had been stored for 5 days at 22 degrees C. When stimulated by ADP and epinephrine as a pair (2 microM each), binding of fibrinogen to platelets was complete within 10-15 min and was not significantly decreased in suspensions from stored concentrates. We also investigated the effect of storage on the glycoprotein IIb-IIa complex, thought to be a specific receptor for fibrinogen on the platelet surface. Binding of a monoclonal antibody specific for this complex (B59.2) to platelet suspensions was unaffected by 5 days of storage. Furthermore, B59.2 inhibited aggregation, secretion, and fibrinogen binding of fresh and stored platelets stimulated with the pair of agents just as it did with single agents. We conclude that storage for 5 days at 22 degrees C impairs the exposure of fibrinogen receptors on platelets in response to ADP or epinephrine when used as single agents, without affecting the glycoprotein IIb-IIIa complex quantitatively. The function of the receptor is normal in response to the pair of agents.