Replacing risk-based early-onset-disease prevention with intrapartum group B streptococcus PCR testing.

OBJECTIVE: To evaluate the effect of a rapid PCR-based group B streptococcus (GBS) test on length of stay in hospital among newborns, antibiotic use, and GBS-early-onset-disease (EOD) incidence. METHODS: We conducted a before and after service evaluation including term deliveries between 1st January and 12th November 2014 (6688 deliveries). Length of stay in the hospital, GBS-EOD incidence and antibiotic use were evaluated. RESULTS: We recorded three confirmed and 74 possible cases of GBS-EOD in Phase 1, and 85 possible cases in Phase 2. In newborns with suspected infection, the introduction of the rapid test was related to a decreased length of stay on the pediatric care unit by 1.16 days (p = 0.01), and an increase in the length of stay on the mother-and-baby ward by 1.11 days (p < 0.001). No increase in antibiotics was noted. CONCLUSION: The introduction of a point of care test was associated with a reduction in length of stay in the pediatric care unit, without an increase in antibiotic use. This test could improve the accuracy of GBS colonization detection, and help to prevent intrapartum transmission as no verified GBS-EOD cases were recorded with the intrapartum PCR algorithm.

An immunohistochemical study of the clearance of apoptotic cellular fragments.

We investigated the distribution and fate of apoptotic bodies during human development and in the adult, using an antibody (M30) that recognizes a neo-epitope formed early in the apoptotic cascade by caspase cleavage of cytokeratin 18. In the fetus, we found extensive accumulation of M30-positive, non-phagocytosed fragments in the red pulp of the spleen, subcutaneous and submucosal vessels, the interstitium of the lung, and the glomerular mesangium of the kidneys. In the liver, M30-immunoreactive fragments were found inside macrophages in the sinusoids. The number of these fragments and the intensity of the immunostaining increased with the gestational age of the fetus. In the adult, M30-positive fragments were barely detectable in normal tissues. However, many pathological situations, including both chronic degenerative processes and metastatic cancer, were associated with accumulation of M30-positive fragments in the red pulp of the spleen. In the liver and kidney, no fragments could be detected. Remarkably, 13 of the 16 patients with metastasized cancer showed pronounced accumulation of M30-positive fragments containing hematoxylin-reactive material in the red pulp of the spleen. In the non-cancerous cases, such DNA-containing fragments were only seen in 9 of 94 cases. The results show that when apoptotic activity is high, as during development in the fetus or during metastasis and other pathological processes in the adult, the phagocytic clearance of apoptotic bodies can be overloaded. These apoptotic fragments then accumulate in the spleen. The visual detection of apoptotic fragments is concluded to reflect increased cell turnover.

The patterns of expression of an apoptosis-related CK18 neoepitope, the bcl-2 proto-oncogene, and the Ki67 proliferation marker in normal, hyperplastic, and malignant endometrium.

Expression of a neoepitope on cytokeratin 18, recognized by the monoclonal antibody M30, is an early indicator of apoptosis in epithelial cells. The aim of this study was to determine the equilibrium between apoptosis (M30), anti-apoptosis (bcl-2), and proliferation (Ki-67) in different endometrial conditions. Paraffin-embedded samples (n = 107), representing proliferative endometrium (18), secretory endometrium (19), postmenopausal endometrium (15), disordered proliferative endometrium (6), simple hyperplasia (12), complex hyperplasia (8), and endometrial adenocarcinoma (29), were evaluated immunohistochemically. The indirect streptavidin-biotin-horseradish peroxidase technique, with 3-amino-9-ethylcarbazole as the chromogen, was used to visualize the reactions. Proliferative endometrium showed high bcl-2 and Ki-67 expression levels with no M30. In the secretory phase, the balance was tipped in favor of M30 with a decrease of bcl-2 and Ki-67. Postmenopausal endometrium revealed high Ki-67 and bcl-2 expression levels and no M30. In complex hyperplasia, M30, bcl-2, and Ki-67 showed increased expression. In endometrial carcinoma, an increasing reactivity for M30 and Ki-67 was seen as the grade progressed. bcl-2 reacted weakly and only in grade 1 cancer. Immunohistochemistry facilitates the study of the expression of proteins related to cyclic endometrial activity. Interruption of these cyclic events is associated with specific disturbances in the expression patterns of these proteins.

Apoptosis, bcl-2 expression, and proliferation in benign and malignant endometrial epithelium: An approach using multiparameter flow cytometry.

OBJECTIVE: Disturbances in the regulation of cell proliferation and differentiation play an important role in the formation of neoplastic lesions. Consequently, abnormalities in apoptosis regulation may contribute to this process. Expression of a neoepitope on cytokeratin 18, unmasked by an early caspase cleavage event and recognized by the novel monoclonal antibody M30, is an indicator of early epithelial cell apoptosis. The purpose of this study was to evaluate the quantitative relation among apoptosis (M30), cell persistence (bcl-2), and proliferation (S-phase fraction; SPF) in malignant and benign endometrium. METHODS: Using multiparameter DNA flow cytometry on 54 formalin-fixed paraffin-embedded samples from benign (proliferative, secretory, inactive, and hyperplastic endometrium) and malignant (grades 1-3 endometrial adenocarcinoma) endometrial tissue, bcl-2 expression and M30 reactivity were assessed together with the SPF in the cytokeratin-positive epithelial cells. RESULTS: Benign cyclic endometrium showed a relatively high bcl-2 expression and low M30 reactivity in the proliferative phase whereas in the secretory phase this relation was inverse. In endometrial hyperplasia the expression of bcl-2 was increased compared to that in secretory and postmenopausal endometrium, but still below the level of proliferative samples. The expression of M30 also increased compared to normal proliferative endometrium but did not reach the level of endometrium in the secretory phase of the menstrual cycle. In cancer the expression of bcl-2 decreased with the progression of differentiation grade. For M30 expression this relation was inverse. Overall there was a significant increase of M30 reactivity in cancerous compared to hyperplasia and normal cyclic endometrium. CONCLUSION: Transition of endometrial epithelium from hyperplasia to cancer seems to involve both increased apoptosis and decreased bcl-2 expression. Flow cytometric evaluation of M30 and bcl-2 expression levels, with the SPF, in currettage specimens from postmenopausal patients complaining of bleeding provides a quantitative assessment of endometrial apoptosis, anti-apoptosis, and proliferation. Further studies are needed to determine the relationship among these three processes as indicators of the biological behavior of gynecological tumors.

Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis.

A neo-epitope in cytokeratin 18 (CK18) that becomes available at an early caspase cleavage event during apoptosis and is not detectable in vital epithelial cells is characterized. The monoclonal antibody M30, specific for this site, can be utilized specifically to recognize apoptotic cells, which show cytoplasmic cytokeratin filaments and aggregates after immunohistochemistry with M30, while viable and necrotic cells are negative. The number of cells recognized by the antibody increases after induction of apoptosis in exponentially growing epithelial cell lines and immunoreactivity is independent of the phosphorylation state of the cytokeratins. The generation of the M30 neo-epitope occurs early in the apoptotic cascade, before annexin V reactivity or positive DNA nick end labelling. In a flow cytometric assay, the majority of the M30-positive cells appear in the 'apoptotic' subG1 peak. Tests with synthetic peptides define positions 387-396 of CK18, with a liberated C-terminus at the caspase cleavage site DALD-S, as the ten-residue epitope of M30. This epitope starts at the end of coil 2 of the predicted CK18 structure, at a probable hinge region, compatible with the sensitivity to proteolytic cleavage. The definition of a specific caspase cleavage site in CK18 as a neo-epitope can be used for quantification of apoptotic epithelial cells with immunocytochemical techniques and is applicable to both fresh and formalin-fixed material.

Distribution of TPA and cytokeratins in gastrointestinal carcinomas as revealed by immunohistochemistry.

The presence and distribution of tissue polypeptide antigen (TPA) were assessed in gastrointestinal carcinomas of different origin, morphology and degree of differentiation. Immunocytochemistry was employed, using the PAP technique on formalin-fixed, paraffin-embedded material and compared with the results obtained with antibodies to cytokeratins. Like cytokeratins, TPA was a reliable marker of epithelial differentiation and showed tissue distribution patterns similar to cytokeratins, as revealed by antibodies with broad-range cytokeratin immunoreactivity. In most carcinomas, TPA-specific immunostaining was less intense than in non-neoplastic tissue. No direct relationship between intensity of TPA staining and morphological degree of differentiation and proliferation was found. TPA staining was most pronounced at the periphery of the cells. In stratified epithelium, i.e. oesophageal mucosa, basally located cells exceeded superficial cells in TPA immunoreactivity in contrast to the cytokeratin antibodies which decorated the more superficially placed cell layers. TPA and cytokeratin staining patterns were similar in neoplastic and non-neoplastic gastric, intestinal mucosa, as well as in biliary tract epithelium. Antral and cardial mucoid glands of the stomach as well as gastric carcinomas of the pylorocardial type remained unstained with both types of antibodies. Similar staining with TPA and cytokeratin antibodies was also observed in pancreatic and liver tissue. In this study, hepatocytes were, although weakly, stained by TPA antibodies and an identical staining was found with benign and malignant hepatocellular neoplasms. Ductal and ductular TPA-staining was most conspicuous and so was the immunoreactivity of cholangiocellular carcinomas. A comparison between TPA and cytokeratins was also made by immunoblotting which revealed immunoreactivity of antibodies to TPA with cytokeratin polypeptides of different species (man, mouse) and organs (epidermis, liver), particularly with the cytokeratin component 8 of human liver and the related component A of mouse liver. The significance of this finding is uncertain until the pertinent epitopes have been revealed by monoclonal mapping of the components which exhibit similar molecular weights by SDS polyacrylamide gel electrophoresis.

Distribution of tissue polypeptide antigen (TPA) in normal human tissues: Immunohistochemical study on unfixed, methanol-, ethanol-, and formalin-fixed tissues.

The distribution of tissue polypeptide antigen (TPA) was studied in unfixed, methanol-, 95% ethanol-1% acetic acid (EA)-, and formalin-fixed paraffin-embedded sections of all adult human tissues using an indirect immunoperoxidase method. The specific staining patterns were virtually identical in unfixed and alcohol-fixed tissues, but in formalin-fixed tissues this similarity was found only after fixation for up to 24 hr and pretreatment with protease for 15 min. Although prolongation of formalin fixation beyond 48 hr increasingly diminished the TPA reactivity, TPA could still be demonstrated in tissues fixed in formalin for up to 6 months. TPA was found to be a cytoplasmic constituent of almost all adult human duct and cavity lining, simple, and stratified epithelia. TPA was not demonstrated in epidermis, renal proximal convoluted and testicular tubules, basket-like myoepithelial cells, nor in most glandular acini, including hepatocytes and pancreatic acinar cells. The TPA staining was also negative in all non-epithelial tissues, including lymph nodes and bone marrow. The well-defined epithelial distribution and the comparable demonstrability in differently preserved tissues make TPA a useful tool for the identification of cells of epithelial character.

Differential immunocytochemical localization of tissue polypeptide antigen (TPA) and carcinoembryonic antigen (CEA) in breast cancer.

The presence of tissue polypeptide antigen (TPA) and carcinoembryonic antigen (CEA) in serial sections of 44 breast carcinomas, one case of neurofibrosarcoma, and one case of fibroadenoma was demonstrated by immunocytochemistry using the indirect peroxidase technique. The neurofibrosarcoma was negative for TPA and CEA. All 44 specimens of breast cancer were positive for TPA and/or CEA. In 18 cases the staining was strongly positive for TPA and CEA. The overall concordance of staining was 43%. TPA was more prominent than CEA in 52% of the tumors while CEA was more prominent in 7%. This is in relative agreement with the values for TPA and CEA in serum from patients with breast cancer, where TPA was higher than CEA in 41% and CEA higher than TPA in 9%. The finding of a difference between and within tumors indicated that TPA and CEA may be synthesized during partly different phases of the proliferative cycle. Assay of TPA and CEA in cytological specimens and in serum should be of clinical interest.

Specificity and basis of the tissue polypeptide antigen.

TPA is an antigenic molecule which is generally present in human carcinoma tumors. Proliferating cells of tumor or normal origin produce and release TPA. Chemical characterization has shown TPA to be an unbranched peptide chain with an apparent Mr of 4.3 X 10(4) and a frictional ratio of 2.7. The electrophoretic mobility is close to that of beta 2-globulin, the IP is 4.5, and the sedimentation constant is 4.5S. The amino acid sequence of the specific determinant is known and has been synthetically produced. Antibodies to TPA obtained by affinity chromatography with BrCN-activated Sepharose, charged with isolated TPA, are responsible for reactivity with TPA by hemagglutination, radioimmunoassay, immunoelectrophoresis, immunodiffusion in gel, and immunocytochemistry. TPA is nonspecies specific and can be found down to fishes. In cell culture (HeLa) perinuclear formation of TPA is seen early in the S phase. After cell division TPA is externalized leaving the cell free of visible TPA. At this stage a rise of TPA in the cell culture medium is observed. These findings together with extensive clinical studies and use of TPA have led to the hypothesis that TPA is related to proliferative activity in general. Since cancer is based on proliferation of cells, and the genes of cancer cells are not different from those of other cells, it could be helpful to use TPA as a monitor of cell proliferation in the further search for local and general causes of proliferation. This can be done by looking for TPA in body fluids by radioimmunoassay and at the cellular level by immunocytochemistry.

Detection of tissue polypeptide antigen in salivary glands and salivary gland tumors. An immunohistochemical study.

Tissue polypeptide antigen (TPA) was analyzed immunohistochemically in parotid gland tissue. This antigen, which is generally regarded as a proliferative antigen, was detected in the ductal system of the normal parotid gland. Parotid gland tumors were analyzed as well: pleomorphic adenomas; cystadenolymphomas; adenoid cystic carcinomas, and mucoepidermoid tumors. TPA could be found in distinct parts of every kind of tumor. However, apart from the TPA-positive cells, negative cells could be observed. The implications of these investigations are discussed.

Immuno-histochemical localization of tissue polypeptide antigen (TPA) and carcino-embryonic antigen (CEA) in breast cancer. A comparative study.

The presence of Tissue Polypeptide Antigen (TPA) and Carcino-embryonic Antigen (CEA) in serial sections of 32 breast carcinomas and one case of neurofibrosarcoma was demonstrated by immunocytochemistry using the indirect peroxidase technique. The neurofibrosarcoma was negative for TPA and CEA. All 32 specimens of breast cancer were positive for TPA and/or CEA. In 12 cases the staining was strongly positive for both TPA and CEA. In other cases either TPA or CEA was more abundant in the tumor. The overall concordance of staining was 58%, which is in relative agreement with the correlation between values for TPA and CEA in serum from patients with breast cancer. The finding that in addition to the difference between tumors, there was a difference within tumors was interesting. This indicated that TPA and CEA may be synthesized during partly different phases of the proliferative cycle.