Spectral imaging of multi-color chromogenic dyes in pathological specimens.

We have investigated the use of spectral imaging for multi-color analysis of permanent cytochemical dyes and enzyme precipitates on cytopathological specimens. Spectral imaging is based on Fourier-transform spectroscopy and digital imaging. A pixel-by-pixel spectrum-based color classification is presented of single-, double-, and triple-color in situ hybridization for centromeric probes in T24 bladder cancer cells, and immunocytochemical staining of nuclear antigens Ki-67 and TP53 in paraffin-embedded cervical brush material (AgarCyto). The results demonstrate that spectral imaging unambiguously identifies three chromogenic dyes in a single bright-field microscopic specimen. Serial microscopic fields from the same specimen can be analyzed using a spectral reference library. We conclude that spectral imaging of multi-color chromogenic dyes is a reliable and robust method for pixel color recognition and classification. Our data further indicate that the use of spectral imaging (a) may increase the number of parameters studied simultaneously in pathological diagnosis, (b) may provide quantitative data (such as positive labeling indices) more accurately, and (c) may solve segmentation problems currently faced in automated screening of cell- and tissue specimens.

Spectral karyotypic study of the HL-60 cell line: detection of complex rearrangements involving chromosomes 5, 7, and 16 and delineation of critical region of deletion on 5q31.1.

Interstitial deletions of the q arm of chromosome 5 have been associated with acute myelogenous leukemia (AML); therefore, accurate identification of rearrangements of this chromosome in a model cell line, HL-60, is important for understanding the critical genes involved in this disease. In this study, we employed a newly developed technology termed spectral karyotyping to delineate chromosomal rearrangements in this cell line. Our study revealed a derivative of chromosome 7 that resulted from translocations of chromosome arms 5q and 16q to 7q; that is, der(7)t(5;7)(?;q?)t(5;16)(?;q?). Interestingly, both chromosomes 5 and 7 were also involved in translocations with chromosome 16 in der(16) t(5;16)(q?;q?22-24) and der(16)t(7;16)(?;q?22-24), respectively. Other notable chromosomal abnormalities that were not previously reported in the HL-60 included an insertion of chromosome 8 in the q arm of chromosome 11, a translocation between chromosomes 9 and 14, and a translocation between chromosomes 14 and 15. In an attempt to define the loss of the 5q31.1 region in HL-60, we performed fluorescence in situ hybridization analysis by utilizing bacterial artificial chromosomes BAC1 and BAC2 that spanned the IL9 and EGR1 gene interval, which was previously shown to be a critical region of loss in AML. We showed that a copy of both BAC1 (spanning the D5S399 locus) and BAC2 (spanning the D5S393 locus centromeric to BAC1) were present in the normal chromosome 5, but a second copy of BAC1 was lost and a second copy of BAC2 was inserted in the der(16)t(7;16) chromosome. Thus, not only was this study the first to use the new 24-color karyotyping technique to identify several novel chromosomal rearrangements in HL-60, but it also narrowed the 5q31.1 critical region of deletion to the region represented by BAC1.

Baboon/human homologies examined by spectral karyotyping (SKY): a visual comparison.

Baboon (Papio hamadryas) metaphase chromosomes were analyzed using spectral karyotyping (SKY), a technique combining fluorescence microscopy, CCD-imaging, and Fourier spectroscopy. Results from a comparison of SKY analyses using probes derived from human chromosomes on baboon metaphases were consistent with the majority of comparative gene mapping data between the two species. These data were also compatible with earlier studies comparing macaque and human chromosomes. Human (HSA) chromosome 2 was homologous to baboon (PHA) chromosomes 12 (HSA 2q) and 13 (HSA 2p), whereas three baboon chromosomes corresponded to two different human chromosomes: PHA 3 to HSA 7 and HSA 21, PHA 7 to HSA 14 and HSA 15, and PHA 10 to HSA 20 and HSA 22. These results support the retained synteny between the Hominidae and Cercopithecidae genomes.

Early (34-56 days from last menstrual period) ultrasonographic measurements in normal pregnancies.

To assess early embryonic growth and development, 361 pregnancies were studied from 34 to 56 days from last menstrual period. All pregnancies had a subsequent successful outcome. Transvaginal ultrasonography was performed using an Acuson 128 x P/10 with a 5-7.5 MHz probe. Gestational sac diameter, embryonic pole length and embryonic heart rates were measured. Embryonic heart rates were determined by M-mode. Gestational sac diameter, embryonic pole length and embryonic heart rate increased linearly relative to gestational age and to each other. Regression equations comparing gestational sac diameter and embryonic pole length as well as comparing embryonic heart rate with gestational sac diameter and embryonic pole length were constructed. To be normal, gestations that have (i) sac diameter of 20 mm and 30 mm should contain at least a 2 mm and 5 mm embryo with embryonic heart rates of at least 75 and 100 beats per min, respectively, and (ii) embryos measuring 2 mm, 5 mm, 10 mm and 15 mm should display embryonic heart rates of at least 75, 100, 120 and 130 beats per minute respectively.

Multicolor spectral karyotyping of human chromosomes.

The simultaneous and unequivocal discernment of all human chromosomes in different colors would be of significant clinical and biologic importance. Whole-genome scanning by spectral karyotyping allowed instantaneous visualization of defined emission spectra for each human chromosome after fluorescence in situ hybridization. By means of computer separation (classification) of spectra, spectrally overlapping chromosome-specific DNA probes could be resolved, and all human chromosomes were simultaneously identified.

Comparison of pulsatility indices on the day of oocyte retrieval and embryo transfer.

Transvaginal ultrasonography with colour blood flow imaging and analysis of impedance to uterine arterial blood flow has been used to provide an index of uterine receptivity for implantation after IVF/embryo transfer. A mean uterine arterial pulsatility index (PI) > 3.0 at the time of embryo transfer predicted 35% of failures to become pregnant. Cryopreserving embryos in non-receptive cycles and transferring them in receptive cycles would be expected to improve pregnancy rates. Earlier decisions regarding embryo cryopreservation can be made if receptive cycles can be predicted at the time of oocyte retrieval rather than at embryo transfer. To assess differences in uterine artery impedance, PI were measured serially in 107 women on both the day of oocyte retrieval and the day of embryo transfer. Mean PI on the day of oocyte retrieval was 2.52 +/- 0.59, and on the day of embryo transfer was 2.78 +/- 0.45. No significant difference was observed when PI determined on the day of oocyte retrieval were compared with PI on the day of embryo transfer. These data suggest that the PI measurement done on the day of oocyte retrieval could substitute for the measurement done on the day of embryo transfer. This would allow prediction of non-receptive endometria earlier in the cycle. Further studies are needed to evaluate whether cryopreservation of embryos and transfer when the uterus is more receptive will increase the implantation rates.

Very early (24-56 days from last menstrual period) embryonic heart rate in normal pregnancies.

To determine embryonic heart rate in early gestations, 426 ultrasonographic examinations from 24 to 56 days from onset of last menstrual period (LMP) were studied. All pregnancies had a subsequent successful outcome. Transvaginal ultrasonography was performed using an Acuson 128 10XP with a 5 MHz probe. Embryonic heart rate was determined by M-mode. No embryonic heart rate was observed prior to 34 days of gestation from onset of LMP (n = 65). At 35 days, two of 13 (15%) pregnancies had cardiac activity, and by 36 days 16 of 19 (82%) pregnancies had cardiac activity. By day 37 from onset of LMP all pregnancies demonstrated embryonic cardiac activity. From days 34 to 56, mean embryonic heart rate rose from 94 to 166 beats/min. We conclude that embryonic cardiac activity is first apparent at day 34 and should be visible by day 37 in normal pregnancies.

Ultrasonographic predictors of implantation after assisted reproduction.

OBJECTIVE: To assess the utility of endometrial thickness, echogenic endometrial pattern, and uterine artery impedance measured as pulsativity index in predicting implantation. DESIGN: Prospective case-controlled study of infertile patients undergoing assisted reproductive technologies (ARTs). PATIENTS: Four hundred five women undergoing ARTs were studied: 100 women after ET of thawed embryos in natural cycles (frozen ET), 107 women after standardized IVF-ET, 99 women receiving donor oocytes after controlled endometrial development with estrogen and P, and 99 women undergoing IUI with various ovarian stimulation regimens (none, 16; GnRH, 7; clomiphene citrate [CC], 29; hMG, 47). INTERVENTIONS: Transvaginal ultrasonographic examination performed on the day of hCG administration during stimulated cycles; on E2 day 15 during controlled endometrial cycles; and on the day of ovulation during natural, CC, and GnRH pump cycles. MAIN OUTCOME MEASURE: The endometrial thickness, echogenic pattern, and pulsativity index results in 170 conception cycles were compared with 235 nonconception cycles. RESULTS: When conception and nonconception cycles were compared, no difference in mean endometrial thickness and significant differences in the frequency of nonmultilayered pattern and pulsativity index > 3.3 were observed. Forty-seven percent of nonconception and 9% of conception cycles were associated with at least one of these factors. CONCLUSION: Ultrasonic measurements of pulsativity index, resistance index, and echogenic pattern are useful in predicting implantation after assisted reproduction.

Transvaginal chorionic villus sampling.

Chorionic villus sampling (CVS) with either transcervical catheters or transabdominal needles is a widely-accepted method for prenatal diagnosis. However, there exists a small subset of patients in whom sampling is difficult or impossible with either route because of individual anatomic variations. A new method of chorionic villus biopsy has been developed to circumvent these problems, utilizing transvaginal chorionic needle aspiration guided by an intravaginal ultrasound probe. This technique was performed successfully in 15 patients in whom villi could not be obtained by either of the conventional methods. This method now makes CVS possible in essentially all women regardless of their uterine anatomy or placental placement; it may also prove useful for very early chorionic sampling.

Far-field behavior of injection-locked semiconductor laser arrays.

We have developed a computer model of injection-locked gain-guided semiconductor arrays to explain some experimental observations: the creation of a single-lobed far field from a normally double-lobed far field; the increase in the divergence angle of the far-field lobe(s); the shifting of power from one off-axis lobe to the other; and the regression back to a double-lobed far field when the ratio between array power and injection power becomes too large. The model is then extrapolated to look at properties of the index-guided case: inefficient end-element injection but an on-axis single-lobed center-element injection.