Rapid, automated assay for progesterone on the Abbott AxSYM analyzer.

We describe an automated assay for progesterone (P4) in human serum and plasma with the Abbott AxSYM random-access immunoassay analyzer. In this one-step competitive assay, P4 immobilized onto latex microparticles competes with sample P4 for binding to a conjugate of alkaline phosphatase (AP) and anti-P4 antibody. Total CVs ranged from 3.4% to 8.2% in multiple precision studies conducted according to the 20-day NCCLS EP5-T protocol. The detection limit (zero calibrator + 2 SD) was 0.10 microg/L across 36 experiments. Values for diluted samples were 83-116% of expected. Recovery of P4 added to serum specimens was 92-115%. Cross-reactivities with 43 natural and synthetic steroids were 0-6.3%. No significant interference was detected from bilirubin, protein, erythrocytes, hemoglobin, triglycerides, or cholesterol. In a multisite correlation study, AxSYM P4 results compared well with results from a commercial RIA method (n = 1156; r = 0.976; slope = 1.03; y-intercept = 0.04). Assay throughput is >80 tests per hour in batch mode, 60 tests per hour with mixed load list configurations.

Neuroendocrine carcinomas of the stomach: a clinicopathologic evaluation.

BACKGROUND: The purpose of this study was to determine by immunocytochemistry the relative incidence and clinicopathologic characteristics of neuroendocrine carcinomas of the stomach. METHODS: Sections from paraffin blocks from 81 patients who had undergone resection of carcinomas of the stomach were immunostained with a battery of neuroendocrine differentiation markers and with A-80, a marker of exocrine differentiation. The clinical and pathologic data of the 12 patients diagnosed with neuroendocrine carcinomas of the stomach were analyzed. RESULTS: The 10 men and two women ranged from 53 to 81 years of age (median, 69 years). Procedures performed included distal subtotal gastrectomy in eight patients and total gastrectomy in four patients. Pathologic stages were stage I, one patient; stage III, four patients; and stage IV, seven patients. Metastatic sites included regional nodes, 11 patients; liver, four patients; and bone, one patient. Adjunct treatment included multiagent chemotherapy plus radiotherapy, four patients; and only radiotherapy, one patient. Eleven patients died of disease 1 to 27 months after diagnosis with an overall median survival of 15 months. Three groups of neuroendocrine carcinomas were identified based on immunostaining patterns. These included pure neuroendocrine carcinomas, two patients; neuroendocrine carcinomas with occasional exocrine cells, three patients; and mixed neuroendocrine-exocrine carcinomas, seven patients. CONCLUSIONS: (1) The relative incidence of neuroendocrine differentiation in carcinomas of the stomach is higher than is generally recognized. (2) Neuroendocrine gastric carcinomas behave aggressively and display numerous structural and functional similarities with their colonic, extrahepatic biliary tract, and pulmonary counterparts.

Elevated plasma platelet-derived growth factor B-chain levels in cancer patients.

Platelet-derived growth factor (PDGF) is produced by a variety of normal and tumor cells in vitro. We have developed an enzyme-linked immunosorbent assay for the detection of the B-chain of PDGF. This assay can reliably detect 0.1 ng/ml of homodimeric recombinant PDGF B-chain and does not cross-react with recombinant PDGF-AA, epidermal growth factor, basic fibroblast growth factor, or transforming growth factor-beta. Citrated plasma from 72 control individuals had a PDGF B-chain (PDGF-B) level of 0.32 +/- 0.14 ng/ml (mean +/- SD) with a range of 0.10-0.69 ng/ml. The plasma platelet factor 4 (PF4) level was 97 +/- 70 ng/ml, with a range of 34-363 ng/ml. Citrated plasma was obtained from 131 cancer patients, and plasma PDGF-B was elevated in 19 (15%) of the patients. Both PDGF-B and PF4 were elevated in 14 (11%) of these patients, consistent with a platelet source of PDGF-B. In 5 patients (4%), however, PDGF-B was elevated and PF4 was not elevated compared to the control group. This last group of patients may have a tumor-derived source of PDGF-B which could be important in autocrine or paracrine growth stimulation of the tumor cells.

Cross validation of cluster analysis using immunostained multi-tissue tumour block slides.

We assayed the panel of SCLC MAbs using multi-tissue tumour block (MTTB) slides obtained from Dr. Hector Battifora (City of Hope Hospital, Duarte CA). MTTB slides contain up to 100 different formalin-fixed tumour tissue specimens and can be immunostained with as little as 50 microliters of antibody solution. In this study, the MAbs in the SCLC panel were used to stain slides from a MTTB comprised of eight normal, ten SCLC, ten squamous cell, ten adenocarcinoma and five undifferentiated lung cancer tissues. Many MAbs in the SCLC panel failed to immunostain the lung MTTB whereas many others showed significant immunoreactivity. Of those MAbs that stained SCLC tissue, none was found to be specific; these MAbs also stained NSCLC tissues or normal lung tissues. Some MAbs in the panel immunostained SCLC and NSCLC tissues, but were also reactive to normal lung tissue as well as other normal tissue specimens. A major advantage of immunostaining MTTBs with a panel of MAbs is that we were able to compare the immunoreactivity of the MAbs on a total of 128 different tissues requiring only 100 microliters of antibody solution using only two MTTB slides per MAb. Although this study was preliminary and certain technical problems in assembling the MTTB as well as optimising the immunostaining procedure for handling 98 or more MAbs simultaneously remain, the MTTB technique remains promising.

Measurement of murine IgG and IgM concentrations in the SCLC MAb panel; effects of concentration on sensitivity and specificity.

Sensitive, solid-phase enzyme immunoassays for detecting murine IgM and IgG were developed and used to quantitate immunoglobulin concentrations in the SCLC MAb panel. Among the IgM MAbs, most contained between 10-75 micrograms ml-1. At least three IgM MAbs, however, contained less than 2 micrograms ml-1 and six contained greater than 75 micrograms ml-1. Two of the three IgM's containing less than 2 micrograms ml-1 immunoglobulin were found to cluster with the negative controls. Among the IgG MAbs, the majority contained between 10-100 micrograms ml-1 Ig. At least five of the IgG MAbs contained greater than 100 micrograms ml-1; and six were less than 2 micrograms ml-1. Three of the MAbs containing less than 2 micrograms ml-1 IgG clustered with the negative controls. Many of the panel members containing greater than 50 micrograms ml-1 antibody were found to give nonspecific immunostaining on tissues and cell lines. Often this nonspecific immunostaining was eliminated when these MAbs were diluted. Although only a minority of the panel members contained very high or very low concentrations of antibody, the data highlight the inherent difficulties that may result, in part from this variable and suggest that efforts be made to normalise the Ig concentrations of panel members in future workshop panels.

Immunohistochemical features of giant cell carcinoma of the lung: patterns of expression of cytokeratins, vimentin, and the mucinous glycoprotein recognized by monoclonal antibody A-80.

Giant cell carcinoma of the lung (GCCL) is an uncommon and extremely aggressive variant of lung cancer. Characteristic microscopic findings include marked pleomorphism, aggregates of mononucleated or multinucleated giant cells (or both), a general lack of architectural cohesiveness, extensive necrosis, and endocytosis by the giant cells. Although the epithelial character of GCCL has been confirmed by a number of studies, controversy persists as to whether it represents a variant of poorly differentiated adenocarcinoma or of squamous carcinoma. Histochemical studies for mucosubstances have yielded variable and conflicting results. This report describes conventionally fixed and processed samples from 10 cases of GCCL studied with a panel of monoclonal antibodies (Mabs) recognizing different cytokeratin polypeptides (AE1, AE3, AE1/AE3 cocktail, and CAM 5.2), vimentin, and Mab A-80, the last of which binds to a mucinous glycoprotein associated with exocrine differentiation. All 10 cases of GCCL reacted with all cytokeratin Mabs; the extent and intensity of the reaction varied notably. All cases stained strongly and diffusely with Mab AE1 and AE1/AE3, the reaction was less extensive and weaker with CAM 5.2. Significantly, 2 cases reacted focally with Mab AE3. Nine cases reacted extensively and intensely with the vimentin Mab, often showing prominent paranuclear globular profiles. All cases reacted with Mab A-80; the reaction was often strong, but the extent was variable. Findings indicate that all GCCL are indeed cytokeratin positive but that most express polypeptides toward the low-molecular weight end of the spectrum; a small subset also expresses heavier polypeptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical evaluation of neoplastic and non-neoplastic breast diseases with Mab A-80.

Five hundred breast tissue samples from 404 cases were immunostained with A-80, a murine IgM Mab that recognizes a mucinous glycoprotein associated with exocrine differentiation. Samples included 196 primary breast carcinomas, 30 breast carcinoma metastases, 118 fibrocystic disease (FCD), and a further group of 84 samples of FCD from cases known to have breast carcinoma. These samples represented a broad spectrum of common and rare variants of carcinoma and FCD. Samples of fibroadenomas, lactating adenomas, cystosarcoma phylloides, gynecomastia, and normal breasts were similarly studied. The vast majority of carcinomas, 203/212 (95.7%) were immunoreactive; staining varied in extent and intensity, and was virtually unrelated to histologic type and to the presence or absence of recognizable glands. In samples including in-situ and infiltrating ductal or lobular carcinoma, reactivity was frequently stronger in the infiltrating components. No significant difference in reactivity between primary and metastatic carcinomas was noted. Of the group of 118 FCD, 27 were negative whereas 91 showed focal and weak staining. Seventy-two/84 FCD with associated carcinoma were immunostained; in 13 of those 72, staining was strong and extensive. Fibroadenomas, lactating adenomas, gynecomastia, and normal "resting" and lactating breast samples stained focally or not at all. Our findings indicate that Mab A-80 is an excellent immunohistochemical marker for the overwhelming majority of breast carcinomas whereas it marks weakly or not at all the majority of benign neoplasms and normal breast. Moreover, Mab A-80 recognizes a subset of FCD that includes proliferative variants associated with an increased incidence of carcinoma, and FCD in association with carcinoma. Questions regarding rare breast carcinomas that do not react with Mab A-80 remain unclear; yet, we believe that Mab A-80 is a highly promising marker of malignant and dysplastic breast epithelium.

Mucin glycoproteins as tumor markers.

Extensive biochemical studies have shown that mucin tumor antigens have a range of molecular sizes from 200 to greater than 1000 kDa. The molecular size of mucin antigens can be dramatically affected by the source and method of purification. Mucin antigens vary from 24 to 80% in carbohydrate content and their density is usually greater than 1.40 g/ml. Galactose and N-acetyl glucosamine are the predominant sugar residues in many mucins, whereas mannose is usually present in low levels or absent. The amino acids serine, threonine, alanine, glycine, and proline are abundant in mucins. An O-glycosidic linkage between the carbohydrate and protein of mucins is the most common linkage encountered. The gene encoding the core peptide for at least one mucin tumor marker, HMFG, has been identified, sequenced, and expressed. These findings may lead to a better understanding of the multiepitope nature of mucin tumor markers. The advent of hybridoma technology has yielded several monoclonal antibodies that have been used to identify the presence of tumor-associated mucins in the sera of cancer patients. Elevated levels of mucin antigens have been found in the serum of most patients with advanced adenocarcinomas. Many studies have shown that tumor-associated markers are useful in monitoring patients following cancer treatment. Clinically useful immunoassays have been developed for monitoring patients with ovarian, breast, and pancreatic adenocarcinomas. Although individual mucin tumor markers show limited utility in detecting early adenocarcinoma, recent studies using multiple mucin markers have suggested that early detection, at sensitivities greater than 50%, can be achieved.

Expression of a new mucin-type glycoprotein in select epithelial dysplasias and neoplasms detected immunocytochemically with Mab A-80.

We studied by immunocytochemistry 573 tissue and 106 cytologic samples of human tumors, non-neoplastic proliferative lesions and normal tissues with the monoclonal antibody (Mab) A-80 that recognizes a mucinous glycoprotein from the colon carcinoma cell line LS-174T. The spectrum of benign and malignant breast lesions was studied as were epithelial tumors of the colon, stomach, pancreas, lung, salivary glands, thyroid, prostate, kidney, endometrium, skin and mesothelium; non-epithelial tumors included lymphomas, melanomas, gliomas, meningiomas, and sarcomas of soft tissue and bone. With a single exception, breast carcinomas regardless of histologic type were reactive while few fibroadenomas stained weakly and focally. In fibrocystic disease, the presence and intensity of the reactivity paralleled the severity of the epithelial proliferation, e.g. staining was strong in foci of severe or atypical hyperplasia, borderline lesions and carcinomas in situ; apocrine metaplasia stained often but less strongly. Barrett's mucosa, colonic polyps and most gastric and colonic carcinomas stained regardless of glandular features while small cell neuroendocrine carcinomas did not. Adenocarcinomas of the pancreas and lung, and a subset of large cell lung carcinomas reacted whereas neuroendocrine carcinomas of those sites did not. Carcinomas of endometrium, ovary and prostate reacted variably whereas thyroid and renal carcinomas and mesotheliomas were either negative or weakly reactive despite the presence of glands. Lymphomas, skin adnexal tumors, nevi, schwannomas, melanomas, gliomas and sarcomas generally did not react but occasional A-80-positive cells were seen in rare sarcomas and meningiomas. Immunostaining patterns in cytologic specimens were similar to the aforementioned. We conclude that Mab A-80 is an excellent marker for breast carcinomas, and for certain proliferative forms of fibrocystic disease that may precede or be associated with carcinomatous transformation. In colonic, pulmonary and gastric carcinomas, staining with Mab A-80 revealed exocrine features regardless of the absence of glands whereas in renal and thyroid carcinomas and in mesotheliomas staining was focal and weak or absent irrespective of glandular features. We suggest that Mab A-80 is a very promising immunolabel for select exocrine carcinomas, and for some of the dysplasias that may precede their development; its ease of application on tissue sections and cytologic specimens should broaden its usefulness.

Molecular characterization of the epitope in prostate and breast tumor-associated PR92 antigen.

In our previous report, monoclonal antibody PR92 has defined prostate- and breast tumor-associated PR92 antigen. The molecular nature of PR92 antigen, especially the epitope involved in specific interaction with PR92 monoclonal antibody, is described. PR92 antigen was purified from the cell extract or tissue culture medium of prostate cancer cell line DU145 by means of monoclonal antibody-coupled Sepharose 4B affinity chromatography, followed by a Sephacryl S-500 chromatography. Physical and chemical characterization, coupled with high-performance liquid chromatography, determined that PR92 antigen is a glycoprotein with a molecular weight of about 470,000, comprising repeating subunits of about 44,000. Sialic acid was found to form a critical part, while D-galactose and N-acetylgalactosamine were also involved, in the epitope structure. PR92 antigen is rich in serine, threonine, proline, glycine, and alanine and poor in aromatic amino acid residues. The carbohydrate moieties may be predominantly O-linked to polypeptide chains which contribute directly or indirectly to maintain the integrity of the epitope. Elucidation of the molecular nature of PR92 antigen may help understand the mechanism of shedding into the body fluids during tumor progression.

Purification and immunochemical characterization of ascitic fluid glycoproteins containing certain tumor-associated and blood group antigen markers.

Ascitic fluids from patients with various types of cancer were screened for the CA 19-9 and CA 125 tumor-associated antigenic activities. Two fluids exhibiting the highest activities were tested for their binding to various lectin-Sepharose columns resulting in both being bound best to wheat germ agglutinin (WGA) Sepharose. The WGA column eluate of one fluid was further chromatographed by HPLC and three peaks were obtained with approximate molecular weights of 3.65 MDa, 664 kDa and 330 kDa, of which only the largest fraction contained the CA 19-9 activity. The fluids were also fractionated on a Sephacryl S-400 column with most of the activity being present in or near the void volume. Monoclonal antibodies were used to demonstrate that the purified glycoproteins also contained the blood group A determinant, the four Lewis determinants Le(a), Le(b), Le(x) and Le(y), and the sialylated-Le(x) determinant, while other antibody analyses failed to detect other blood group and/or carbohydrate sequence determinants. Some of the blood group expressions could be separated from the CA 19-9 and CA 125 active glycoproteins by adsorption with various lectins other than the WGA.

Immunohistochemical analysis of colon carcinomas applying exocrine and neuroendocrine markers.

Eighty colon carcinomas reflecting the histologic spectrum were studied immunohistochemically; their epithelial characteristics had been established by demonstrating cytokeratin polypeptides. Paraffin sections were immunostained with monoclonal antibody (Mab) A-80 that recognizes a mucin-like glycoprotein related to exocrine differentiation. Sequential sections were immunostained with neuroendocrine (NE) differentiation antibodies: NSE, human chromogranin A, serotonin, somatostatin, substance P and VIP. Twenty-one/80 carcinomas immunoreacted exclusively with Mab A-80; these included adenocarcinomas with variably defined glands, colloid, "solid", and linitits plastica carcinomas. Eleven/80 carcinomas immunoreacted only with antibodies to NE markers. Twenty-nine/80 carcinomas of histologically variable patterns expressed both exocrine and NE antigens. A notable group of 19 adenocarcinomas immunostaining with Mab A-870 included a minority NE cell subpopulation. We tentatively conclude that given a limited battery of immunoprobes, colon carcinomas comprise 4 groups: 1) pure exocrine carcinomas, 2) pure NE carcinomas, 3) mixed exocrine and NE carcinomas, and 4) exocrine carcinomas with occasional NE cells. Thus, phenotypically mixed exocrine and NE carcinomas comprise the largest group while the second largest group exhibited exclusively features of exocrine phenotype. Preliminary clinical correlative data indicate that pure NE colon carcinomas behave more aggressively than their exocrine counterparts; moreover, colon carcinomas containing a NE subpopulation, even if small, also seem to behave worse than their counterparts without an NE subpopulation.

A monoclonal antibody against neuron-specific enolase. Immunohistochemical comparison with a polyclonal antiserum.

There is skepticism about the value of antisera to neuron-specific enolase (NSE) for immunohistochemical identification of neural and neuroendocrine differentiation in neoplasms, because of reports of detection of NSE, in a large percentage of nonneuroendocrine neoplasms. By immunohistochemical methods, the authors compared a monoclonal antibody to NSE (Mab NSE) with a heterologous antiserum to NSE (Het NSE) on 348 samples of tumors of diverse histogenesis. They studied 93 neural and neuroendocrine tumors and 255 nonneuroendocrine, nonneural tumors. The Mab NSE was slightly less sensitive but clearly more specific than the Het NSE in recognizing neural and neuroendocrine differentiation. Only 2% of the nonneuroendocrine, nonneural tumors reacted positively with Mab NSE; in contrast, 20% of the same tumors were positive with the Het NSE. Moreover, intense nonspecific staining was frequent with Het NSE, which often rendered interpretation difficult. Because of its superior specificity, the Mab NSE used in this study is more valuable than the heterologous antiserum as a diagnostic reagent in tumor diagnosis.

Evaluation of a new enzyme-linked immunoassay for terminal deoxynucleotidyl transferase in haematologic malignancies.

We have evaluated a new solid phase enzyme immunoassay (EIA) for detection of terminal deoxynucleotidyl transferase (TDT). The EIA is greater than 100 times more sensitive than previously used tests for enzyme activity. In 284 clinical specimens of human peripheral blood and bone marrow, the EIA detected TdT antigen in 97% of peripheral blood and 100% of bone marrow samples that were positive for enzymatic activity. The excellent sensitivity and specificity of this new test suggests that it can be used in clinical situations where quantitative TdT measurements are desired.

A new solid-phase immunoassay for terminal deoxynucleotidyl transferase: analysis of TdT antigen in cells, plasma, and serum.

A solid-phase immunoassay for terminal deoxynucleotidyl transferase has been developed using a primary antibody-coated polystyrene bead and secondary antibody conjugated with horseradish peroxidase. The immunoassay was compared with assays for enzyme activity and detection of antigen with immunofluorescence using cells from peripheral blood and bone marrow from patients with leukemia or lymphoma. In each instance, the solid-phase immunoassay correlated correctly with cellular samples judged to be positive by other tests. However, the level of detection of terminal transferase antigen in plasma or serum of patients with leukemia did not reflect accurately the level of terminal transferase in neoplastic cells. The solid-phase immunoassay was greater than 100-fold more sensitive than conventional assays for enzyme activity, rendering it potentially useful for quantitatively monitoring terminal transferase in patients with leukemia.

A specific enzyme-linked immunosorbent assay (ELISA) for the determination of human C5a antigen.

An enzyme-linked immunosorbent assay has been developed to detect human C5a antigen. This ELISA methodology has been shown to be a highly sensitive technique capable of detecting C5a antigen concentrations below 10 ng/ml. The microELISA technique used in this study is specific for human C5a and C5a des arg (C5a antigen) but not for human C5. Conditions to establish sensitivity and specificity are outlined in ths report.

Purification of human C5a des arg by immunoadsorbent and molecular sieve chromatography.

Human C5a des arg was isolated from complement-activated serum by immunoadsorption followed by Sephadex G-75 chromatography. C5a des arg obtained by this 2-step procedure was shown to be immunologically identical to C5a des arg purified by a conventional multi-step method, homogeneous on SDS-polyacrylamide gels, and biologically active. Although this technique yields approximately the same amount of C5a des arg/liter of activated serum as that obtained by conventional methods, its simplicity and relative rapidity make it a practical alternative.

Chemotaxis under agarose utilizing human serum depleted of C-5 derived peptides.

An immunoabsorbent column was made with antibody to trypsinized human C5. This column removed the chemotactic activity from zymosan-activated serum as well as from C5a des-arg-enriched fractions. Anti-trypsinized C5-absorbed human serum was substituted for unabsorbed human serum in the chemotaxis under agarose system. This resulted in significantly reduced random neutrophil migration with a negligible effect on C5a des-arg or FMLP-directed migration. The results indicate that much of the random migration observed in the chemotaxis under agarose system is due to C5-derived peptides present in normal human serum.