A method of processing first-trimester chorionic villous biopsies for cytogenetic analysis

Chorionic villous biopsy is emerging as a technique for obtaining fetal cells for prenatal diagnosis in the first trimester of pregnancy. Chromosome analysis has been performed on small villous biopsies using either direct harvests of uncultured cells or after culturing villous tissue. Here, we describe a method where both techniques can be used simultaneously; from a single villous biopsy, GTG-banded chromosomes of improved morphology are obtained from direct preparations and from cultured villous cells.     

Murine bone marrow culture system for cytogenetic analysis

A mouse bone marrow culture system for examining genotoxicity of agents by first exposing animals in vivo then growing cells in vitro is presented. This assay can also be used for in vitro and/or for the in vivo and in vitro comparative cytogenetic studies. The protocol involves culturing of approximately 1,000,000 nucleated cells obtained from mice tibia and femora in 5 ml of Ham's F-12 medium containing 20% fetal bovine serum, 10% whole uterus extract from pregnant mice and 1% penicillin-streptomycin. The use of flasks and mouse uterus extract for culturing are important steps for higher mitotic yield. The addition of 20 microM BrdU for 24 h helps in the differentiation of sister chromatids for sister-chromatid exchange (SCE) analysis. Cyclophosphamide, given to mice through intraperitoneal injection, induced significant dose-related SCEs in culture. Trinitrofluorenone, a direct-acting mutagen, caused dose-related SCEs in in vitro bone marrow cell culture.     

The effect of refrigeration of bone marrow and peripheral blood on cytogenetic analysis

Summary Bone marrow samples from patients with various hematologic disorders were stored at 4° C for up to 5 d before the establishment of a 24-h culture. We tested various factors, including storage time, colony stimulating factor, and methotrexate in an effort to improve metaphase and chromosome quality. Cytogenetic findings for various hematologic diseases were compared in a total of 201 cultures. Cold storage for up to 3 d did not seem to adversely affect the number of mitoses or the quality of chromosome banding when cells were cultured in a system that used both colony stimulating factor and methotrexate. In samples studies in parallel, clonal abnormalities were noted as frequently in cells stored in the cold as in those processed directly. This work was supported by contract DE-AC02-80EV10328 and by grant CA 16910 from the National Cancer Institute, Bethesda, MD.     

The use of foetal ovarian stromal cell culture for cytogenetic diagnosis. Stromal ovarian culture cytogenetic diagnosis

Some studies have been carried out to analyze human female first meiotic prophase. Most of them use samples from foetuses collected after legal interruption of pregnancy. In some cases, a control population is needed and foetuses aborted for non-chromosomal reasons are used. The assumption of these samples as being euploids could perhaps represent an error. In this article, we describe an easy methodology to certify the euploidy of foetal ovarian tissue using an one-week somatic culture. Using this protocol, we have obtained a primary culture in 88.2% of the studied cases, material usable for being karyotyped in 93.3% of the cases, and a cytogenetic diagnosis was performed in 100% of these cases. Finding the same karyotype in cultured cells in cases in which we had a prenatal cytogenetic diagnosis has validated the technique, and in applying this protocol we have been able to check our prophase meiotic-study control population.     

Sediment cytology in bone biopsies

The cytologic examination of smears prepared from the sediment of biopsy specimen fixatives ("sediment cytology") was used to study 70 bone lesions biopsied with a suspicion of malignancy. The smears were adequately cellular in most cases and showed good morphologic preservation; some contained fragments of tissue. Cytology was able to identify the smears from the 47 malignant lesions as malignant, but was not always able to identify the histologic type. While the osteoclastomas, Ewing's sarcomas and metastatic carcinomas were accurately diagnosed, the osteogenic sarcomas could only be identified as sarcomas and the scanty smears from chondrosarcomas only permitted a diagnosis of malignancy. The latter was also true for soft tissue lesions and lymphoma involving the bones. The 12 benign lesions yielded less cellular specimens and were more difficult to cytologically diagnose. The 11 inflammatory lesions were identified as nonmalignant. While this simple technique of sediment cytology can provide an early diagnosis for bone lesions, the final diagnosis requires the histopathologic study of the actual biopsy specimen.     

The use of cytogenetic tools for studies in the crop-to-wild gene transfer scenario

Interspecific hybridization in plants is an important evolutionary phenomenon involved in the dynamics of speciation that receives increasing interest in the context of possible gene escapes from transgenic crop varieties. Crops are able to cross-pollinate with a number of wild related species and exchange chromosome segments through homoeologous recombination. In this paper, we review a set of cytogenetic techniques that are appropriate to document the different steps required for the stable introgression of a chromosome segment from a donor species (i.e., the crop) into a recipient species (i.e., the wild). Several examples in hybrids and derivatives are given to illustrate how these approaches may be used to evaluate the potential for gene transfer between crops and wild relatives. Different techniques, from classical chromosome staining methods to recent developments in molecular cytogenetics, can be used to differentiate genomes and identify the chromosome regions eventually involved in genetic exchanges. Some clues are also given for the study of fertility restoration in the interspecific hybrid forms.     

The utilization of interphase cytogenetic analysis for the detection of mosaicism

This study describes a method for defining mosaic aneuploidy by interphase cytogenetics based on statistical limits established from control specimens. Fluorescence in situ hybridization (FISH) has been used to detect the number of copies of specific chromosomes in interphase nuclei from placental tissues of diploid controls and mosaic placentas. FISH was performed using probes D7Z1/D7Z2, D9Z1, D10Z1, and D18Z1, all purchased from Oncor, Inc. Statistical analysis of data obtained from diploid controls was used to determine the one-sided upper reference limit and corresponding 95% confidence interval for the proportion of cells with one and three signals for each of the probes used. The one-sided upper reference limits established the lower levels of monosomy and trisomy detectable using each of the four probes. These statistical parameters were then used to interpret the results obtained by FISH applied to the study of term placentas for the confirmation of prenatally diagnosed chromosomal mosaicism.     

Microwave histoprocessing of bone marrow trephine biopsies

Summary In recent years, the microwave oven has been increasingly used in the pathology laboratory for processing of tissue for diagnostic purposes with a remarkable reduction in processing time and also reports of excellent morphology and immunohistochemistry. We evaluated some of these processes on post mortem bone marrow trephine biopsies and describe a novel way of processing these biopsies in the microwave oven.     

The use of needle biopsies for radiobiological assessment of oxygen levels in KHT-C tumors.

Hypoxia affects the sensitivity of cells to radiation. Hence there is considerable interest in the development and assessment of techniques for measuring oxygen levels. In the work described here, we explore the use of tumor needle biopsies (fine needle aspirates) in an assay that is standard in the field of radiation biology: the paired survival assay. We found that needle biopsies are a feasible option for estimating cell survival when conducting this assay, and that the variability in cell survival between tumors was greater than that between different biopsies from the same tumor. Using this technique, we then compared measurements of tumor hypoxia using the paired survival assay and the growth delay assay in the same individual tumors. We found a significant correlation between these two techniques.     

The use of repetitive DNA in cytogenetic studies of plant sex chromosomes

The structure of sex chromosomes in plants was analyzed by fluorescent in situ hybridization (FISH) with repetitive DNAs. FISH probes were successfully obtained from DNA libraries that were amplified from microdissected sex chromosomes. Some probes hybridized to the subtelomeric regions, where many kinds of repetitive DNAs are located with intrachromosomal similarity of their repeat units rather than interchromosomal similarity. For example, FISH with the subtelomeric repetitive sequence can easily show the location of the pseudoautosomal region (PAR) on the X chromosome of Silene latifolia. The other probes were localized on the interstitial region of the sex chromosomes. The interstitial region contains chloroplast DNAs or neighboring sequences of the internal telomeres, suggesting insertion or translocation occurred during differentiation of the sex chromosome. These data are very informative for understanding the structure of the plant sex chromosomes and their evolutionary process.     

Accuracy of touch imprint cytology of image-directed breast core needle biopsies

OBJECTIVE: To evaluate the accuracy of touch imprint (TI) cytology of image-directed core needle biopsy (CNB) specimens of nonpalpable breast lesions. STUDY DESIGN: Fifty-two consecutive CNBs from 44 patients were performed under mammographic or ultrasound guidance. Air- dried TIs of CNBs were stained with Diff-Quik. TI cellularity was considered adequate if six or more ductal cell groups were identified. CNBs and TIs were interpreted in a blinded fashion. RESULTS: TI cellularity was adequate in 37/52 (71%) cases, including 17/20 carcinomas and 20/32 benign lesions. Among 17 carcinomas, TIs were positive in 12, suspicious in 4 and atypical in 1. One case of lactational change was suspicious on TI, and 5/8 fibroadenomas were atypical. No benign lesions were called "carcinoma" on cytology. When lesions categorized as "carcinoma" or "suspicious" were considered positive and those classified as "atypical" or "benign" were scored as negative, TI sensitivity and specificity were 94% and 95%, respectively. When the "atypical" cases were grouped with the positive cases, TI sensitivity was 100%, with 75% specificity. CONCLUSION: With satisfactory cellularity, TIs of CNBs are highly accurate. When immediate evaluation of CNB specimens is important, TIs can potentially decrease the number of biopsy passes required and provide preliminary diagnoses.     

Technical note: The use of geographical information systems software for the spatial analysis of bone microstructure

Geographic information systems (GIS) software is typically used for analyzing geographically distributed data, allowing users to annotate points or areas on a map and attach data for spatial analyses. While traditional GIS-based research involves geo-referenced data (points tied to geographic locations), the use of this technology has other constructive applications for physical anthropologists. The use of GIS software for the study of bone histology offers a novel opportunity to analyze the distribution of bone nano- and microstructures, relative to macrostructure and in comparison to other variables of interest, such as biomechanical loading history. This approach allows for the examination of characteristics of single histological features while considering their role at the macroscopic level. Such research has immediate promise in examining the load history of bone by surveying the functional relationship between collagen fiber orientation (CFO) and strain mode. The diversity of GIS applications that may be utilized in bone histology research is just beginning to be explored. The goal of this study is to introduce a reliable methodology for such investigation and our objective is to quantify the heterogeneity of bone microstructure over an entire cross-section of bone using ArcGIS v 9.3 (ESRI). This was accomplished by identifying the distribution of remodeling units in a human metatarsal relative to bending axes. One biomechanical hypothesis suggests that CFO, manifested by patterns of birefringence, is indicative of mode of strain during formation. This study demonstrates that GIS can be used to investigate, describe, and compare such patterns through histological mapping.     

A technique to obtain fibroblast cells from skin biopsies of living bats (Chiroptera) for cytogenetic studies

We developed a procedure to obtain fibroblasts from bat skin. A small fragment of the ear is removed under ether anesthesia. This material is then cut up into small pieces and cultured in standard cell culture media. Very good quality chromosome preparations for cytogenetic studies are obtained in about three weeks. Secondary cultures can be used for other biological studies. This procedure does not require sacrificing the animals.     

A semi-automated cytogenetic analysis system

Because cytogenetic evaluations involve a number of time-consuming steps, we have employed semi-automated procedures to speed up and facilitate the analysis of chromosomal aberrations in industrial workers. The system contains mechanisms for automatic focus, slide search, metaphase spread detection and relocation, centering, and X-Y coordinate memory. The equipment automatically locates and then displays a cell on a television screen; computer measurements are performed in a 0.1 second. An electronic light pen is used to mark and edit chromosomes directly on the television monitor. Preliminary studies have shown significant time-cost reduction compared with conventional procedures, and the results of analyses on the television monitor are as reliable as those from microscopic investigations.