Cervical trophoblasts for non-invasive single-cell genotyping and prenatal diagnosis.

OBJECTIVE: We aimed at developing a method to recover trophoblastic cells from the cervix through a completely non-invasive approach and obtaining a genetic proof of their fetal nature implying that they can be used for non-invasive prenatal diagnosis (NIPD). METHODS: We studied obstetrical samples from 21 pregnant women between 8 and 12 weeks of gestation scheduled for chorionic villus sampling or undergoing elective termination of pregnancy. A cytobrush was used to extract cells from the external parts of the cervix and transferred to 10 ml of preservative solution. Cells were layered on filters with 8 microns pores using the ISET system (Isolation by SizE of Tumor/Trophoblastic cells) and stained. Putative fetal cells were collected by single cell laser-assisted microdissection and identified as fetal or maternal cells by Short Tandem Repeat genotyping. NIPD was blindly performed on 6 mothers at risk of having a fetus with Cystic Fibrosis or Spinal Muscular Atrophy. RESULTS: Trophoblastic cells were recovered from all tested cervical samples with a frequency of 2-12 trophoblasts per 2 ml. NIPD was blindly obtained and verified in 6 mothers at risk of having a fetus with Cystic Fibrosis or Spinal Muscular Atrophy. DISCUSSION: Although larger confirmation studies are required, this is the first report providing a solid proof of principle that trophoblasts can be consistently and safely recovered from cervical samples. Since they are a source of pure fetal DNA, i.e. fetal DNA not mixed with maternal DNA, they constitute an ideal target to develop NIPD of recessive diseases, which is a technical challenge for methods based on cell free DNA.

The significance of an elevated serum lysozyme value in acute myelogenous leukemia with eosinophilia.

According to criteria established by the French-American-British (FAB) classification, a diagnosis of acute myelomonoblastic leukemia (FAB M4) is based on the presence of 20% bone marrow monocytes or a serum lysozyme level that exceeds the reference value by three times. Reported here is a case of acute myelogenous leukemia with eosinophilia and a cytogenetic inversion of chromosome 16 (inv 16) that lacks morphologic, cytochemical, and immunophenotypic features of monocytic differentiation, but which is associated with an elevated serum lysozyme value. The authors used an immunoelectron microscope to localize lysozyme to both normal and abnormal eosinophil granules, in addition to the secondary granules of myeloid precursors and monocytes. This enzyme could not be demonstrated within the myeloblasts of the patient studied. Postfixation with osmium tetroxide greatly reduced the staining intensity within the crystalloids of normal eosinophils, but only minimally affected that of monocytes, neutrophils, normal eosinophil granule matrix, and the abnormal granules of the leukemic eosinophils. These results demonstrate that lysozyme is present in both normal and leukemic eosinophils and that elevation of serum lysozyme in patients with acute myelogenous leukemia with eosinophilia is not a reliable indicator of monocytic differentiation. Furthermore, an occasional case of acute leukemia with inv 16 is classifiable as acute myelogenous leukemia with differentiation (FAB M2).

[Non-invasive prenatal diagnosis of cystic fibrosis]. / Diagnostic prénatal non invasif de la mucoviscidose.

Cystic fibrosis (CF) is a frequently fatal autosomal recessive inherited disease affecting around one in 3000 newborns in France, the carrier frequency varying from one in 20 to one in 40 subjects depending on the geographical area. The disease is caused by a chloride channel defect that is attributable to mutations in the gene that encodes the CF transmembrane conductance regulator (CFTR). Approximately, 1200 different mutations have been discovered. Among them, the F508del mutation accounts for 70% of mutated alleles worldwide. Prenatal diagnosis (PND) of inherited monogenic disorders such as CF currently relies on invasive procedures--amniocentesis, chorionic villus sampling (CVS)--which carry a significant risk of miscarriage (from 0.5 to 3%). Several methods have been proposed to enrich circulating fetal cells (CFCs) from blood and use them in PND. However, up to now, no assay has been shown to be reliable enough for routine application in place of the invasive protocols. When combined with laser microdissection, isolation by size of epithelial tumor/trophoblastic cells (ISET) allows mutation analysis of DNA from single cells demonstrated to be fetal (circulating fetal trophoblastic cells [CFTC]) by short tandem repeat (STR) genotyping and uncontaminated with maternal DNA. Application of this protocol to 12 couples at risk of having a child affected by CF has shown, in a blind study, that the new method affords a reliable and safe PND of affected fetus, healthy carrier or normal non carrier fetus. A following prospective blind study has then been performed on 32 couples at risk of having an affected child. For each mother, five or 10 CFTCs have been analyzed with an individual genetic diagnosis performed per CFTC. Results have been obtained in 240 CFTC showing that seven mothers were carrying an affected foetus, with 100% sensitivity and 100% specificity. These results open the way to a multicenter clinical validation trial and to the potential future application of the ISET non invasive approach as a reliable alternative to the invasive PND procedures.

Long-term follow-up after osteotomy for haemophilic arthropathy of the knee.

In this study the long-term value of corrective osteotomy around the knee was evaluated by means of clinical and radiographic parameters. Between 1974 and 1984 we performed 52 corrective osteotomies in the vicinity of the knee on patients affected by haemophilic arthropathy. Forty-two patients (45 osteotomies) were adequately followed-up at an average 11.6 years postoperatively. Using the clinical score of the Advisory Committee of the World Federation of Haemophilia, 38 patients showed a postoperative improvement, five remained clinically unchanged and two showed deterioration. Range of motion of the knee joint did not significantly improve postoperatively. The radiographic Pettersson score showed only a marginal decrease by an average 0.003 points at the time of follow-up. Only one patient needed subsequent joint replacement of both knees, on the left side 13 years after osteotomy and on the right side 8 years after osteotomy. Even in cases of marked radiographic joint destruction, corrective osteotomy shows acceptable long-term clinical results, underlining the feasibility of this management option in the treatment of haemophilic arthropathy of the knee. Although moderate cartilage degenerations in the femoropatellar complex and in the contralateral compartment can be tolerated, this therapy should primarily be contemplated for those patients where damage is unicompartmental and a corresponding axial deviation is found. Particularly the younger patient can benefit from this treatment option in that joint replacement may possibly wholly be avoided or at least postponed to a later stage of life.

Demonstration of human papillomavirus DNA by nucleic acid in situ hybridization in paired histologically abnormal cervical biopsies obtained at the same patient visit.

Fifty-four pairs of cervical biopsies ranging from minimal dysplasia to severe dysplasia were studied for the presence of human papillomavirus DNA by in situ hybridization. Two assays were performed on each biopsy. A 16 hour hybridization was used in one assay, while a 40 hour hybridization was utilized in the second assay. Increasing the hybridization time to 40 hours did not significantly increase the detection rate of HPV compared to the rate found using the 16 hour hybridization. Also, no difference in the detection rate of HPV was found by using one biopsy of the pair over the other biopsy of the pair. However, the performance of a single in situ assay on only one biopsy from each patient significantly underestimated the true prevalence of HPV. A single assay only detected 21/33 (64%) patients with HPV. Implications of multiple testing of all histologically abnormal biopsies is discussed in relation to prospective follow-up studies determining the usefulness of HPV typing in patient management.

Detection of human papillomavirus DNA in genital lesions by using a modified commercially available in situ hybridization assay.

A modified, commercially available DNA-DNA in situ hybridization test that uses biotinylated probes for the identification of human papillomavirus (HPV) DNA types 6/11, 16/18, and 31/33/35 was evaluated. HPV DNA was detected in 314 of 787 (40%) histologically abnormal genital biopsy specimens by using the ViraType in situ assay (Life Technologies, Gaithersburg, Md.), in which the hybridization time was increased from 2 to 16 h. Ninety percent of positive condyloma acuminata specimens contained HPV type 6/11 DNA. The prevalences of HPV DNA for cervical intraepithelial neoplasia I, II, and III lesions by this in situ hybridization test were 42, 54, and 55%, respectively. The combined prevalence of HPV type 16/18 and 31/33/35 DNAs increased with the severity of the lesion, while the prevalence of type 6/11 DNA decreased. HPV type 6/11 DNA was found only in 1 of 16 (6%) positive cervical intraepithelial neoplasia III specimens. HPV type 16/18 and 31/33/35 DNA was detected in 11 of 16 (69%) and 4 of 16 (25%) in situ hybridization-positive cervical intraepithelial neoplasia III specimens, respectively. Thus, the observation that certain "higher-risk" HPV genotypes are associated with upper-grade cervical precancer lesions was confirmed by this commercial hybridization system. In general, the assay was found to be well suited for use in the clinical laboratory. The ViraType in situ procedure modified for a longer hybridization time may be helpful in identifying lesions containing higher-risk HPV strains.