Biomedical subjects

M Nap

Publications and source records attributed to M Nap.

60 records · Page 4Linked to original sources

PubMed1977

Immune complex detection by immunofluorescence on polymorphonuclear leucocytes.

Polymorphonuclear leucocytes (PMN) from patients with systemic lupus erythematosus (SLE) were isolated from defibrinated and heparinized blood. In addition, PMN from a healthy donor were incubated with sera from SLE patients and with sera containing artificially prepared immune complexes of hepatitis B surface antigen (HBsAg) and human anti-HBsAg immunoglobulin (anti-HBs) with well defined variations of the antigen/antibody ratio. To one group of blood samples, 5 mM monoiodine acetic acid (MIAA) was added to block in vitro phagocytosis. The Pmn were examined for the presence of IgG, IgM, and HBsAg by the immunofluorescence technique. PMN from defibrinated blood of SLE patients showed in up to 80% immunoglobulin (Ig)-inclusions. However, addition of 5 mM MIAA reduced the number of Ig-containing PMN to at most 40%, which levels were equal to numbers found in specimens from heparinized blood. Addition of 5 mM MIAA to heparinized blood did not reduce the number of PMN with Ig inclusions. Normal donor PMN isolated from defibrinated, heparinized, and EDT blood showed equal amounts of Ig inclusions after incubation with SLE sera, but none when MIAA had been added. In PMN incubated with HBsAg-anti HBs immune complexes with an antigen antibody ratio between 5 and 0-2, both HBsAg and IgG could be detected. It is concluded that Ig inclusions in PMN from heparinized blood from SLE patients are due to in vivo phagocytosis, presumably of circulating immune complexes. In vitro phagocytosis of Ig from SLE sera by normal donor PMN also suggests the presence of immune complexes. Dependent on the antigen-antibody ratio, artificial HBsAg/anti-HBs immune complexes can be detected by in vitro phagocytosis by PM.

Antigen-Antibody Complex↗

PubMedDate not supplied

Steroid receptor heterogeneity in relation to DNA index in breast cancer: a multiparameter flow cytometric approach on paraffin-embedded tumor samples.

Steroid hormone (estrogen and progesterone) receptor (ER and PR) status at the time of breast carcinoma surgery is used as a marker for hormone dependency to guide adjuvant therapy. In a significant number of cases a discrepancy exists between the detected number of hormone receptors and the response to hormonal therapy. One of the explanations for this could be intratumoral heterogeneity. Our objective was to investigate the heterogeneity of steroid hormone receptor expression in breast cancer by using multiparameter flow cytometry (MP-FCM) on routinely processed formalin-fixed, paraffin-embedded tumors. A series of 232 routinely processed breast carcinomas were analyzed using a recently developed technique for the isolation of single cells from paraffin-embedded material. After dewaxing and rehydrating, 50-microm thick sections were heated for 2 hours at 80 degrees C in a citrate solution. Single-cell suspensions were prepared by a short pepsin digestion. The obtained single-cell suspensions were immunostained simultaneously for cytokeratin and ER or PR. Finally, DNA was stained using propidium iodide, after which the samples were analyzed on a flow cytometer. The fractions of ER- and PR-positive cells were determined in the total, as well as the G0 /G1 fraction of the diploid, and in case of nondiploid tumors, also in the G0 /G1 fraction of the aneuploid cell population. Of 232 cases, 88 (38%) were diploid, 38 (16%) were tetraploid, and 106 (46%) were aneuploid. In the diploid tumors the mean fraction of ER- and PR-positive cells was 81% and 76%, respectively. The ER- and PR-positive fractions in the total cytokeratin-positive fraction decreased significantly in the tetraploid (56% and 55%, respectively) and aneuploid tumors (both 47%, p < 0.0001). When analyzing the ER- and PR-positive fractions separately in the diploid and aneuploid cell populations of the nondiploid tumors, it became apparent that the ER and PR status in the diploid fraction of the tumor was significantly higher than in the aneuploid fraction (p < 0.0001). For the tetraploid tumors the mean ER- and PR-positive fractions were 79% and 76%, respectively, in the diploid fraction, and this decreased to 45% in the aneuploid cell subpopulation. In the aneuploid tumors this decrease was even more drastic: in the diploid cell population the ER- and PR-positive fractions were 66% and 62%, while this was 38% and 39% in the aneuploid population. These findings illustrate clearly the existence of a heterogeneous distribution of ER/PR expression in breast cancer, related to the loss of a diploid DNA index. Because of its objective quantification of subfractions within the same tumor, MP-FCM can be regarded as a superior method compared to more conventional techniques such as immunohistochemistry and biochemistry.

Adult↗

PubMedDate not supplied

Immunohistochemical profiles of 30 monoclonal antibodies against cytokeratins 8, 18 and 19. Second report of the TD5 workshop.

In the first report of the TD5 workshop (TD5-1), the epitope specificities of 30 different monoclonal antibodies against cytokeratins 8, 18 and 19 were determined. This second report presents the immunohistochemical profiles of these antibodies using human appendix and normal skin for evaluation. Each antibody was tested by one or two different laboratories recruited from the Dutch Working Group on Immunohistochemistry and Cytochemistry. Eight different laboratories participated. The histological specimens were pretreated by the participants in three different ways for immunohistochemistry: microwave antigen retrieval in citrate buffer, enzymatic digestion to restore epitope exposure, no specific treatment (untreated paraffin-embedded samples), and tested blindly without knowledge of cytokeratin or epitope specificity of the antibodies at three different concentrations of 50, 10 and 1 microg/ml. Most of the tested antibodies (29/30) were useful in at least one pretreatment method, with microwave antigen retrieval being the most sensitive approach. For some antibodies, very high backgrounds were observed. Furthermore, it can be concluded that 11 MAbs performed well using all three staining protocols, including untreated paraffin-embedded sections. Interestingly, all the antibodies with documented selected specificity towards cytokeratin 8 (i.e. 178, 191, 199, 202 and 206) are reactive with an immunodominant region corresponding to amino acids 340-365 on cytokeratin 8, which evidently is well-suited as target for immunohistochemical interactions. Similarly, three antibodies with the same capacity to react with untreated samples had specificity against cytokeratin 19 (i.e. 179, 197 and 204) in the corresponding region in this filament, i.e. amino acids 311-335, or the KS 19.1 epitope. None of the six antibodies against the other major cytokeratin 19 epitope (BM 19.21) were found useful for immunohistochemistry on untreated samples. The overall conclusions from the present investigation are that all cytokeratin-8-specific antibodies with defined epitope specificities were very useful. Only one of the major two epitopes on cytokeratin 19 seems to be available for efficient immunohistochemistry. Cytokeratin 18 exposes some epitopes outside the immunodominant region reactive with the antibodies 190, 203 and 205 which can be used for untreated samples. The implications of these findings are of significance both for diagnostic histopathology and for the biology of tumor marker epitope expression in tissues.

Animals↗

PubMedDate not supplied

Immunohistology of carcinoembryonic antigen (CEA). Specificity of reagents, choice of control tissues and practical application in surgical pathology.

Immunohistology of CEA has been used now for many years in research and in diagnostic applications. In this congress paper, the need for specificity checks, correct preservation and quality control of antibodies as well as the choice and preservation of control tissues will be discussed and, in addition, some aspects of the interpretation of CEA immunohistology in surgical pathology are illustrated, based upon prospectively collected data from a routine surgical pathology unit. An approach for collection and analysis is demonstrated and the diagnostic importance of CEA expression together with other antigens in the same tumor.

Antibody Specificity↗

PubMedDate not supplied

Immunohistochemistry of CA 125. Unusual expression in normal tissues, distribution in the human fetus and questions around its application in diagnostic pathology.

CA 125 is known as the marker that is most strongly associated with epithelial gynecological tumors. Compared to the number of publications on its use in serum assays, the application in immunohistochemistry is still limited. The availability of many good antibodies that perform well in formalin-fixed paraffin-embedded tissue opens good possibilities for a wider use. Outside the gynecological tract several other structures may react positive for CA 125. Among these are the lung and breast but also the epithelial cells of the conjunctiva and to some extent prostate glandular epithelium. In the fetus reactions can be found in the serosal linings of body cavities but also in the esophagus and skin. In diagnostic pathology CA 125 plays a role in identifying the primary locations of metastatic carcinoma of unknown origin. It is recommended to use CA 125 antibodies not in a solitary setting but in combination with CEA, BRST-2 and Vimentin to discriminate best between the most frequent sites of origin of metastatic carcinoma. Regular analysis of sensitivity/specificity ratios in a balanced population, representing the composition of the patient population seen in daily practice, should be performed to evaluate the position of CA 125 in diagnostic immunohistochemistry.

CA-125 Antigen↗

PubMedDate not supplied

Immunohistochemical detection of P-glycoprotein on frozen and paraffin-embedded tissue sections of normal and malignant tissues.

In this study the reactivity of the monoclonal antibodies (mabs) C 219, C 494 and 4E3 was compared on frozen and paraffin-embedded normal tissues and tumors. On frozen tissues we used an indirect immunoperoxidase method, while on paraffin sections a streptavidin-biotin method without antigen retrieval methods was used. In normal tissues all mabs were reactive with colon epithelium in frozen and paraffin-embedded sections. The bile canaliculi in the liver showed the most extensive reaction with C 219 in frozen sections, and to a lesser extent with C 494 and 4E3. C 219 reacted with the pancreatic acinar and ductal epithelium, whereas C 494 and 4E3 reacted predominantly in the stroma. The kidney showed positivity for all mabs in the collecting ducts and isolated solitary cells were reactive in the spleen. In the skin the eccrine part of the sweat glands was reactive for all mabs in paraffin sections. The lung, prostate and breast were negative for all mabs. Only in paraffin sections of various tissues did the C 494 appear to be reactive with nerve fibers and ganglion cells. Colon cancers were positive for P-170 with all mabs tested. In breast carcinoma C 494 showed positive reactions in 8/26 frozen and 4/22 paraffin sections, while 4E3 was reactive with 20/25 frozen but only with 1/21 paraffin sections. C 219 gave similar results in frozen (22/26) and paraffin (17/26) sections of breast cancer. Ovarian carcinomas were positive with C 494 in 11/20 of the frozen and in 11/15 of the paraffin sections, while 4E3 again reacted more weakly in paraffin (5/15) than in frozen (15/20) sections. C 219 gave positive reactions in all ovarian carcinomas in frozen (20/20) and paraffin sections (14/14). In the tumors, the most intense reaction for all mabs was obtained in the colon, followed by the ovary and breast. Enhanced staining was seen in paraffin sections for mab C 494 in ovarian carcinoma. By demonstrating the presence of both an external and internal epitope on frozen sections, the combined use of 4E3 and C 219 gave complementary information about the expression of P-170.

ATP Binding Cassette Transporter, Subfamily B, Mem↗