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SummaryThe practical utility and diagnostic accuracy of two new rapid, automated and quantitative immunoturbidimetric D-dimer methods have been evaluated in a population of 123 randomly selected patients with suspected VTE. The STA Liatest D-dimer and MDA D-dimer methods are based on the photo-optical measurement of the rate of agglutination of antibody-coated latex particles. The VIDAS D-dimer automated Elisa was used as the reference method. Diagnosis was confirmed in 51 patients (29 PE, 19 DVT, 3 DVT+PE). The immunoturbidimetric methods compared favorably with the VIDAS Elisa as judged from the correlation coefficients of linear regression lines (r = 0.82, MDA vs VIDAS; r = 0.75, STA vs VIDAS) and areas under the curve of ROC plots (VIDAS 0.83; STA 0.83; MDA 0.81). At a discriminant value of 500 ng/mL, all three D-dimer assays showed high sensitivity (96-98%), high NPV (93-97%) and moderate specificity (39-42%). Reproducibility of results around the cut-off is an important aspect of the diagnostic utility of D-dimer assays. CV's of duplicate determinations in this critical zone showed average values of 5.4% and 17.0% for MDA and STA, respectively. These data demonstrate that such rapid and automated latex-based methods for the quantitative measurement of D-dimer hold promise as reliable and cost-efficient approaches for the exclusion of VTE. Prospective patient management studies will be required to confirm this.

The nucleophosmin (NPM) gene is involved in two recurrent translocations in hematological malignancies: t(2;5) (p23;q35) in anaplastic large cell lymphoma (ALCL) and t(3;5)(q25.1;q34-35) in myelodysplasia and acute non-lymphocytic leukemia (ANLL). Using eight YACs encompassing the 5q34-q35 region, we could easily detect these two translocations. In both types of translocation, probable unexpected deletions were also discovered downstream of the breakpoint at 5q35.

Epidermolytic palmoplantar keratosis (EPPK) cosegregates with breast and ovarian cancers in a large French pedigree, raising the possibility that a single genetic mutation might cause these conditions and offering a potential lead to the identification of a hereditary breast/ovarian cancer gene. We have performed linkage analysis and show that the EPPK locus lies on the long arm of chromosome 17 near the type I keratin gene cluster and the proposed breast cancer gene (BRCA1). The type I keratin 9 gene has been partially sequenced in four affected individuals. A single base mutation within the rod domain of the protein cosegregates with EPPK in all affected individuals tested. Although inheritance of this mutation is likely responsible for EPPK, it is unlikely to be the cause of the breast and ovarian cancer.

The practical utility and diagnostic accuracy of two new rapid, automated and quantitative immunoturbidimetric D-dimer methods have been evaluated in a population of 123 randomly selected patients with suspected VTE. The STA Liatest D-dimer and MDA D-dimer methods are based on the photo-optical measurement of the rate of agglutination of antibody-coated latex particles. The VIDAS D-dimer automated Elisa was used as the reference method. Diagnosis was confirmed in 51 patients (29 PE, 19 DVT, 3 DVT+PE). The immunoturbidimetric methods compared favorably with the VIDAS Elisa as judged from the correlation coefficients of linear regression lines (r = 0.82, MDA vs VIDAS; r = 0.75, STA vs VIDAS) and areas under the curve of ROC plots (VIDAS 0.83; STA 0.83; MDA 0.81). At a discriminant value of 500 ng/mL, all three D-dimer assays showed high sensitivity (96-98%), high NPV (93-97%) and moderate specificity (39-42%). Reproducibility of results around the cut-off is an important aspect of the diagnostic utility of D-dimer assays. CV's of duplicate determinations in this critical zone showed average values of 5.4% and 17.0% for MDA and STA, respectively. These data demonstrate that such rapid and automated latex-based methods for the quantitative measurement of D-dimer hold promise as reliable and cost-efficient approaches for the exclusion of VTE. Prospective patient management studies will be required to confirm this.

To establish human renal cell carcinoma (RCC) xenografts for preclinical studies, 55 renal tumors (33 primary and 22 metastatic lesions) were transplanted subcutaneously into severe combined immunodeficient mice. Twenty of 49 evaluable tumors (40.8%) grew with a median latency period of 89 days (36 to 209 days) from the day of engraftment. Tumor growth was stabilized after the fifth passage with a median time between passages of 38 days (19 to 80 days). Tumorigenicity was correlated with the metastatic phenotype of the tumor (54% success rate, p = 0.007) and with reduced survival of patients. Despite a possible evolution of histological features and tumor grading, established RCC xenografts were comparable to parental tumors, as assessed by karyotype and DNA-ploidy analyses. Molecular cytogenetic analysis also revealed specific genetic alterations characterizing distinct RCC types that were constant in parental and corresponding xenografts. In addition, this xenograft model has permitted the selection of minor tumor subclones with a proliferative advantage and minimal overexpressed chromosomal regions. We conclude that severe combined immunodeficient mice are useful recipients for the establishment of long-term RCC xenografts that can be used as valuable tools to evaluate the activity of new therapeutic approaches and to study biological parameters determining in vivo aggressiveness of human RCC.

The International Normalized Ratio (INR) has not lowered the interlaboratory differences in prothrombin time (PT) values to the extent expected, mainly because of the instrument-dependency of the International Sensitivity Index (ISI) and other factors (eg, accurate determination of the ISI, the normal value used in the PT ratio). The procedure (PPC) using plasma calibrants (reference lyophilized plasmas with assigned activity) has been evaluated since 1977 in nine French national external quality assessment surveys (NEQAS) involving approximately 4,000 laboratories and numerous local thromboplastin technique combinations. The PPC was compared with the conventional procedure (using the manufacturer's ISI), and the efficiency of antivitamin K-calibrated (AK Cal) plasmas from patients receiving oral anticoagulants vs artificially depleted plasma calibrants was also evaluated. The PPC efficiently standardized PTs with AK Cal plasmas, reducing interlaboratory variability (eg, coefficient of variation, 12% to 6% for survey 92 D) and reagent-instrument effects. However, AK Cal plasmas have drawbacks, such as limited supply, cost, and batch-to-batch variability. The artificially depleted plasma calibrants were less efficient, but usable if carefully prepared. The value of this simple procedure is that local practices are considered in the determination of PT, thus correcting for coagulometer effects and avoiding use of the manufacturer's ISI and need for a normal control plasma. These large-scale French surveys have demonstrated the validity of PPC through 15 years of experience and have shown that it offers the best compromise available in PT standardization.

Chromosomal assignment and analysis of chimerism of 22 YACs was performed by FISH. Probes were obtained by PCR amplification of the human YAC inserts with Alu primers. Maximum amplification of various inter-Alu elements was obtained when the primer annealing temperature was below the optimal temperature needed for high specificity. In these conditions, yeast DNA contributed to the amplification of various Alu-PCR products and, since strong competition was required for the suppression of all Alu sequences, yeast Alu-PCR products fulfilled this purpose efficiently.

Four recent interlaboratory surveys have been conducted by the French National "Etalonorme" Group of Quality Control in Haematology, involving about 3,800 laboratories working in coagulation. They have been concerned with human lyophilised plasma samples heparinized in vitro either with unfractionated heparins (UFH), or with low molecular weight heparins (LMWH) (Fragmin 89 D, and Fraxiparin 90 A). The doses added simulated therapeutic situations or were on the border-line of over- or underdosage: UFHs 89 B3/B4, 0.14 and 0.22 IU/ml; 89 C3/C4, 0.3 and 0.4 IU/ml respectively; LMWHs: D3-A3, 0.6; and D4-A4, 1.2 IU/ml. Automated partial thromboplastin times (APTT) were prolonged at these high doses of LMWH, due to noticeable residual anti-IIa activity. A relationship between APTT and heparinaemia was observed with both types of heparin; sensitivities of cephalin reagents to heparin were also noted, but they were different for UFH compared to LMWH. Dispersion on APTT results was still high (CV: 12-23% for the UFH samples, 12-19% for the four LMWH samples). Dispersion on heparin determinations was higher for UFH than for LMWH, even for lower anti-IIa activity, but they involved more heterogeneous assays. The anti-Xa activity for LMWH samples was mainly determined using clotting assays (70% of the laboratories) with larger dispersions (CV: 32-37%), than those observed on amidolytic assays (CV: 28%); however, these amidolytic assays were performed in 90% of the laboratories with only two reagents available from a single manufacturer. The clotting techniques, more than the colorimetric assays, seemed to underestimate the high heparinaemias.(ABSTRACT TRUNCATED AT 250 WORDS)

Hybridization of a poorly immunogenic tumor cell with an allogeneic cell was performed in order to improve tumor immune response; several variants derived from one hybrid tumor cell were studied. We compared the immunogenicity of these variants and their allogeneic and syngeneic class I antigen expression before and after IFN gamma treatment. Allogeneic class I antigens were weakly expressed in all variants; IFN treatment enhanced their expression similarly in both immunogenic and nonimmunogenic variants. Syngeneic class I antigen expression differed among variants: IFN treatment induced changes in their expression which corresponded to a posttranscriptional event and which could, at least partly, explain the modifications observed in their immunogenicity.

When injected in admixture with tumor cells, the peptidoglycan extracted from Bacillus megaterium inhibited the growth of a chemically induced fibrosarcoma in syngeneic rats. In some instances, the tumor growth was totally suppressed. Animals rejecting mixed inocula exhibited a tumor-specific immunity. Peptidoglycan was not cytotoxic for tumor cells; it rendered macrophages nonspecifically cytotoxic.

The adjuvant and mitogenic properties of bacterial peptidoglycans (PG) of different structures have been compared. The PG from Bacillus megaterium and Staphylococcus aureus were adjuvant-active in the guinea-pig, and mitogenic for guinea-pig and mouse lymphocytes. On the other hand the PG from Micrococcus lysodeikticus and Corynebacterium poinsettiae exerted neither adjuvant nor mitogenic activities. Mitogenic PG were selective B mitogens. They were mitogenic for splenocytes from Nude mice and inactive towards T-cell-enriched populations. They were mitogenic in both germ-free mice and in the C3H/HeJ strain which is unresponsive to lipopolysaccharide.

Cytotoxic responses mediated by effector cells stimulated in vivo were studied after ip injection in mice of peptidoglycans extracted from gram-positive bacteria. A comparison was done between Staphylococcus aureus peptidoglycan (PGS), which possessed an antitumor effect, and Micrococcus lysodeikticus peptidoglycan, which was ineffective against tumors. Both peptidoglycans induced similar effects on the modulation of T-cell cytotoxic response. Both were able to stimulate splenic and peritoneal nonspecific cytotoxicity against YAC-1 lymphoid tumor cells, but only PGS could induce cytotoxicity and cytostasis against solid tumor target cells.

Spleen cells from WAG rats bearing methylcholanthrene-induced sarcomas were cytotoxic for homologous tumour cells and for some cell lines derived from other syngeneic tumours. Each tumour induced its own pattern of cross-reactive cytotoxicity. This cross-reactivity was not apparent by in vivo rejection tests. In tumour-bearing rats, specific cytotoxic cells could not be separated from cross-reactive cytotoxic cells. The effector cells did not adhere to plastic surface, they were retained neither on nylon wool nor on anti-Ig columns and were devoid of Fc receptors. After tumour excision the cells displaying specific cytotoxicity had the same properties and thus were probably T cells. In contrast, the cells exhibiting cross-reactive cytotoxicity were lost after passage through a nylon wool column or after the removal of Fc-receptor-bearing lymphocytes. These findings suggest that cross-reactivity could result from antibody-dependent cell-mediate cytotoxicity directed against shared antigens.

The in vitro influence of twelve antibiotics (representing the principal classes: β-lactam group, aminoglycosides, polypeptidic antibiotics, antimitotics, . . .) on the ADP-induced Platelet Aggregation, the Screen Filtration Pressure test and the liquid phase Electrophoretic Mobility (E. M.) has been studied. According to the results formerly obtained on the interference Penicillin G-platelets, the authors have mainly investigated the β-lactam group (Penicillin G, Ampicillin, Carbenicillin, Cefalotin, Metampicillin, Methicillin, Oxacillin, . . .). This group seems particularly interesting as an antiaggregating effect has been found out for most of them; a parallel decrease of the E. M. has been generally observed.In attempts to elucidate the mechanism of physiochemical interaction with the membrane, the competitive effect of phospholipases and Penicillin G has been studied. The results seem to point out an interference of this antibiotic with the membrane phospholipids; this could be in accordance with the rheological data of Padefield and Kellaway. The interference with some other membrane groups has also been considered (carboxyl, phosphates . . .).An in vivo study has also been realized on patients massively perfused with some of these antibiotics (especially Penicillin G, Carbenicillin . . .).(This work was supported by Grant of the DRME (Biological Section).)

The Organon Teknika-General Diagnostics Coag A Mate X2 (CAM X2), Ortho Koagulab 40A, MLA Electra 700 (E 700), were simultaneously evaluated following the same protocol, on the same samples. Compared with the former photo-optical-based automated devices, these newer advanced instruments present a high throughput in fibrin endpoint detection, permitting fibrinogen determination with reliable results; they also present a higher degree of automatism, greater flexibility in use and easy mastering, allied to elaborated expression of results. Statistical comparison shows equivalent performance characteristics. Repetabilities are excellent for Prothrombin Time (CV: 1-2.5%) with 5 thromboplastins and for the prothrombin complex factors assays: they are also good for Activated Partial Thromboplastin Time (APTT) with 4 reagents (CV: 0.5 to 3%) provided that kaolin be not used on the KLO and E700; the same limitation exists for specific factors assays. Fibrinogen assays display good results (CV: 3-4% and 6-8%) respectively for times of 11 sec. (/=/ 2.4 g/l) and 23 sec. (/=/ 1 g/l). Standard linearities are generally excellent whatever the test. The correlation with results obtained on other devices is good (0.96 less than R less than 1). The most fully automated is the KLO with reliable system of sample delivery, automatic calibration and quality control function. The E700 is the slowest instrument, especially for APTT and the least adapted to large homogenous series, but its original loading system with binary coded cuvettes makes it interesting for emergencies and mixed tests. Controls and adjustments of the pumptubing are particularly required on the CAM X2 which is, on the other hand, the quickest instrument with its 4 photo-optical cells, well adapted to handling a large series of samples.

The antitumor effect of peptidoglycans of various structures extracted from different gram-positive bacteria was studied, on chemically induced fibrosarcomas, in C3H/He, C57BL/6, and (C57BL/6 X C3H/He)F1 mice. When given sc admixed with tumor cells, only some peptidoglycans (those extracted from Bacillus megaterium and Staphylococcus aureus) enhanced tumor resistance in syngeneic and semiallogeneic hosts, whereas other peptidoglycans (those extracted from Micrococcus lysodeikticus and Corynebacterium poinsettiae) possessed no antitumor effect. When tumor cells were given ip, administration of peptidoglycans by the same route was either without effect on tumor growth or it induced tumor enhancement. Enhancement could be observed with all of the peptidoglycans tested. The antitumor effect when given sc and the ability to stimulate the proliferation of B-lymphocytes were shared by the same two peptidoglycans, while the other two peptidoglycans were devoid of both activities. It appears that these biological activities depend on the structure of the peptide moiety of peptidoglycans and that mitogenic and antitumor responses are stimulated by similar structures.

Peritoneal nonspecific cytotoxicity was stimulated by ip injection into C57BL/6 or (C57BL/6 X C3H/He)F1 mice of Staphylococcus aureus peptidoglycan (PGS), which possessed an antitumor effect, and of Micrococcus lysodeikticus peptidoglycan (PGM), which was ineffective against tumors. The natural killer (NK) cell populations elicited by both peptidoglycans had the same phenotype: Thy 1.2+, T200+, Ly 5.1+, Qa5+, and Ly 2.2-. In vivo treatment with sheep anti-mouse beta interferon serum abrogated this effect. The fluids from phorbol myristate acetate-stimulated murine EL 4 thymoma cells enhanced this particular NK activity, and thus PGM-induced effector cells became able to kill solid tumor cells. In conclusion, both PGS and PGM elicited the same particular subset of NK cell populations, and the peptidoglycan structure can play an important role in the intensity of this response.