Using the cavitation collapse time to indicate the extent of histotripsy-induced tissue fractionation.

Histotripsy is an ultrasonic tissue ablation method based on acoustic cavitation. It has been shown that cavitation dynamics change depending on the mechanical properties of the host medium. During histotripsy treatment, the target-tissue is gradually fractionated and eventually liquefied to acellular homogenate. In this study, the change in the collapse time (t col) of the cavitation bubble cloud over the course of histotripsy treatment is investigated as an indicator for progression of the tissue fractionation process throughout treatment. A 500 kHz histotripsy transducer is used to generate single-location lesions within tissue-mimicking agar phantoms of varying stiffness levels as well as ex vivo bovine liver samples. Cavitation collapse signals are acquired with broadband hydrophones, and cavitation is imaged optically using a high-speed camera in transparent tissue-mimicking phantoms. The high-speed-camera-acquired measurements of t col validate the acoustic hydrophone measurements. Increases in t col are observed both with decreasing phantom stiffness and throughout histotripsy treatment with increasing number of pulses applied. The increasing trend of t col throughout the histotripsy treatment correlates well with the progression of lesion formation generated in tissue-mimicking phantoms (R 2 = 0.87). Finally, the increasing trend of t col over the histotripsy treatment is validated in ex vivo bovine liver.

In vivo transcostal histotripsy therapy without aberration correction.

This study investigates the in vivo therapeutic capabilities of transcostal histotripsy without using aberration correction mechanisms and its thermal impact on overlying tissues. Non-invasive liver treatments were conducted in eight pigs, with four lesions generated through transcostal windows with full ribcage obstruction and four lesions created through transabdominal windows without rib coverage. Treatments were performed by a 750 kHz focused transducer using 5 cycle pulses at 200 Hz PRF, with estimated in situ peak negative pressures of 13-17 MPa. Temperatures on overlying tissues including the ribs were measured with needle thermocouples inserted superficially beneath the skin. Treatments of approximately 40 min were applied, allowing overlying tissue temperatures to reach saturation. Lesions yielded statistically comparable ablation volumes of 3.6 ± 1.7 cm(3) and 4.5 ± 2.0 cm(3) in transcostal and transabdominal treatments, respectively. The average temperature increase observed in transcostal treatments was 3.9 ± 2.1 °C, while transabdominal treatments showed an increase of 1.7 ± 1.3 °C. No damage was seen on the ribcage or other overlying tissues. These results indicate that histotripsy can achieve effective treatment through the ribcage in vivo without requiring correction mechanisms, while inducing no substantial thermal effects or damage to overlying tissues. Such capabilities could benefit several non-invasive therapy applications involving transcostal treatment windows.

Renal ablation by histotripsy--does it spare the collecting system?

PURPOSE: The feasibility of histotripsy (transcutaneous nonthermal mechanical tissue fractionation) was previously demonstrated in an in vivo rabbit renal cortex model. We explored the spectrum of histotripsy bio-effects on different tissue types in an in vitro porcine kidney model. MATERIALS AND METHODS: Using an 18 element focused annular array ultrasound system we performed histotripsy treatments in 5 in vitro porcine kidneys, targeting 7 cortical volumes and 17 tissue volumes bridging the cortex, medulla and/or collecting system. Treated areas were observed using ultrasound. In 5 lesions methylene blue was infused into the collecting system to evaluate the preservation of collecting system integrity. Kidneys were sectioned and examined grossly for evidence of tissue fractionation, ie the presence of histotripsy paste, or fixed in formalin and prepared for histological analysis. RESULTS: Histotripsy of renal cortical tissue created tissue defects in the cortical area treated. Histotripsy targeting the renal collecting system, medulla and renal cortex resulted in tissue fractionation in the area of the cortex, intermediate damage in the medulla and minimal damage to the collecting system. CONCLUSIONS: There is a differential histotripsy treatment effect when comparing renal cortical tissue to renal collecting system. There is no significant architectural disruption of the renal collecting system after histotripsy. This differential effect is a notable finding that may prove useful in future planning of ablative treatments for renal tissue.

Microwave-induced thermal imaging of tissue dielectric properties.

A new imaging method, microwave-induced thermal imaging (MITI), was developed to differentiate tissue based on thermal and dielectric properties. Image contrast depends on temporal strain in tissue, which was determined by one-dimensional speckle tracking using a phase-sensitive, correlation-based technique. The underlying mechanisms were analyzed and experimental results on biologic tissue agreed well with theoretical predictions. Because of its strong contrast between water-bearing and lipid-bearing tissue, the technique may enhance existing intravascular ultrasound (IVUS) imaging systems to identify vulnerable arterial plaque.

Comparison of methods for erythroblast selection: application to selecting fetal erythroblasts from maternal blood.

BACKGROUND: Many methods have been employed to obtain fetal cells from maternal blood for prenatal diagnostics, but there has been little work done that compares the efficacy of different methods. This study presents a comparison of two commonly used methods for selecting erythroblasts with selection directly from whole blood. METHODS: Erythroblasts were isolated from maternal blood by either differential lysis or density separation, followed by selection with an antibody to the transferrin receptor. These methods were compared with antibody selection directly from whole blood. The total yield of erythroblasts was determined for each method. RESULTS: Red cell lysis is not recommended because the lysis step cannot be well controlled. Density separation followed by antibody selection works well. However, a faster and simpler method, antibody selection directly from whole blood using Immunicon Ferrofluid and magnetic separators, works as well and has the potential to yield even more cells. CONCLUSIONS: Considering the need for a simple and quick method for selecting fetal cells from maternal blood, we suggest selection directly from whole blood.

A novel method for depositing erythroid cells onto glass slides for fetal cell analysis.

BACKGROUND: We have developed a method for selecting erythroblasts from blood, the first step toward identifying fetal cells in maternal blood for diagnostic purposes. Because the selection method results in a large number of positive cells, we needed to develop new methods to deposit the cells onto slides and to modify in situ hybridization procedures to enable detection of fetal cells. METHODS: We utilized Nunc flaskettes to increase the slide surface area available for cell deposition. The ability of erythroid lineage cells to adhere to several surface modifications was examined. In situ hybridization methods were tested to find the best approach that is compatible with these cell preparations. RESULTS: The best glass slide coating for erythroid cells was found to be an antibody to glycophorin A, a red cell surface antigen. We were able to get excellent in situ hybridization signals in cells on flaskettes by modifying fixation and pretreatment parameters. CONCLUSIONS: The methods described here appear to be the best way of attaching a large number of erythroid lineage cells to slides and of detecting them by in situ hybridization.

Inconsistency of fetal trophoblast cells in first trimester maternal peripheral blood prevents non-invasive fetal testing using this cell target.

We have investigated whether maternal peripheral blood from the first trimester of pregnancy is a reliable source of identifiable trophoblast cells. The cells were enriched from 30 ml of venous blood, with multiple antibodies shown previously to enrich trophoblasts and a new cocktail based on known trophoblast surface features. Three different magnetic solid phases were tested to enrich trophoblasts, and both positive and negative cell enrichment strategies were examined. The cells were identified as trophoblast by morphology coupled with immunocytochemistry to co-localize cytokeratin with one of three IGF-II, PAI-1 or hPLH proteins or by in-situ hybridization with a mixture of 50 oligos directed to eight different expressed genes, alpha-HCG, IGF-II, PAI-1, HASH2, hPLH, p57(KIP2), PP5, H-19. While these tools worked beautifully in chorionic villi cell/sprout preparations and tissue sections, we could not detect and identify any trophoblasts in maternal peripheral blood even if the maternal peripheral blood was drawn 5-20 min following termination of pregnancy or from individuals maintaining the pregnancy. Based on our own experience and that of some reports in the literature, trophoblasts do not appear to be a viable candidate for fetal screening using maternal peripheral blood as the source. It is important to note that while trophoblast deportation is a biological phenomenon that has been described repeatable, they do not provide a means to perform prenatal genetic diagnosis.

Enrichment and identification of fetal trophoblast cells from first trimester maternal cervical lavage and uterine blood specimens.

First trimester prenatal diagnosis of fetal aneuploidies is an active area of research despite years of disappointing data employing maternal peripheral blood samples. To remedy this situation we have investigated other first trimester maternal specimens attempting to find a consistent fetal cell source. Using our previously established positive enrichment procedure along with a commercially available depletion method, fetal trophoblast cells were identified employing immunocytochemistry using an antibody cocktail or by using mRNA in-situ hybridization employing a cocktail of trophoblast specific probes. Fetal origin of positively identified cells was verified using interphase fluorescent in-situ hybridization (FISH) for X and Y-chromosomes. Artificial model systems were established that indicated yields of trophoblast cells and allowed the enrichment procedure to be optimized for minimal losses from maternal specimens. We demonstrate herein that blood drawn from maternal vessels near the placental implantation site to be the most consistent source of fetal cells from any first trimester maternal specimen described to date. In addition, a high yield of multinucleated syncytiotrophoblast cells was obtained using a cell depletion strategy to enrich the target cells. The safety of the procedure or even the clinical utility of blood drawn from maternal vessels near the placental implantation site is yet to be demonstrated.

High-density hapten labeling and HRP conjugation of oligonucleotides for use as in situ hybridization probes to detect mRNA targets in cells and tissues.

Oligonucleotides that carry a detectable label can be used to probe for mRNA targets in in situ hybridization experiments. Oligonucleotide probes (OPs) have several advantages over cDNA probes and riboprobes. These include the easy synthesis of large quantities of probe, superior penetration of probe into cells and tissues, and the ability to design gene- or allele-specific probes. One significant disadvantage of OPs is poor sensitivity, in part due to the constraints of adding and subsequently detecting multiple labels per oligonucleotide. In this study, we compared OPs labeled with multiple detectable haptens (such as biotin, digoxigenin, or fluorescein) to those directly conjugated with horseradish peroxidase (HRP). We used branching phosphoramidites to add from two to 64 haptens per OP and show that in cells, 16-32 haptens per OP give the best detection sensitivity for mRNA targets. OPs were also made by directly conjugating the same oligonucleotide sequences to HRP. In general, the HRP-conjugated OPs were more sensitive than the multihapten versions of the same sequence. Both probe designs work well both on cells and on formaldehyde-fixed, paraffin-embedded tissues. We also show that a cocktail of OPs further increases sensitivity and that OPs can be designed to detect specific members of a gene family. This work demonstrates that multihapten-labeled and HRP-conjugated OPs are sensitive and specific and can make superior in situ hybridization probes for both research and diagnostic applications.

Identification of differentially expressed mRNAs in human fetal liver across gestation.

Differential gene expression, with its precise start and stop times, is believed to be critical for the programmed development of new cells and tissues. Within the developing fetus, one tissue of particular interest is fetal liver. This organ undergoes rapid changes in the pathway toward liver development in utero since it is also the major site of hematopoiesis, until bone marrow hematopoiesis predominates. Believing that patterns would emerge from the bi-weekly large-scale inspection of expressed genes in the fetal liver, we employed differential display reverse transcription-polymerase chain reaction (DDRT-PCR) as ourprimary inspection tool. Using DDRT-PCR, we isolated cDNAs differentially expressed throughout fetal liver development and in adult liver. We displayed approximately 25 000 cDNAs from 10 and 24 week fetal liver and adult liver. From this initial screen, we determined that approximately 0.1-1% of the mRNA population undergoes expression changes. We extracted, purified and sequenced 25 differentially displayed cDNA bands. Fourteen cDNAs had similarities to known genes, while 11 cDNAs were not similar to any characterized gene. The differentially expressed cDNAs from known genes present in fetal liver include alpha-fetoprotein, stem cell factor, erythroid alpha-spectrin, 2,3-bisphosphoglycerate mutase, insulin-like growth factor-2, porphobilinogen deaminase and Mac30. The differentially expressed cDNAs present in adult liver but not in 10 week fetal liver were nicotinamide deaminase, human fibrinogen-related protein and alpha-acid glycoprotein. The majority of differentially expressed genes found during this effort appear to be turned on during organogenesis, however, some genes were found that are apparently turned off completely.

Dimethylformamide as an enhancer of cavitation-induced cell lysis in vitro.

Polar solvents, including dimethylformamide (DMF), have been investigated as anticancer drugs, but their potential usefulness is constrained by hepatotoxic side effects. The ability to enhance drug cytotoxicity with ultrasound would be valuable in creating locally intense chemotherapy while minimizing effects peripheral to the treatment site. The effects of continuous wave ultrasound (US) (985 kHz; 0.5-2.5 W/cm2) were evaluated on cultured HL-60 human promyelocytic leukemia cells alone and with a noncytotoxic DMF dose (0.11 M). The cells were insonified in a configuration that created no cell lysis without the introduction of albumin-stabilized microbubbles into the exposure chamber. When microbubbles were introduced, US with bubbles induced cell lysis, and the presence of DMF significantly increased the lysis induced by ultrasound with bubbles. The necessary presence of microbubbles for the DMF-US synergism to occur suggests that a likely mechanism is acoustic cavitation, initiated by the presence of microbubbles as nuclei. Detection of subharmonics confirmed the presence of cavitation, and cell lysis was well correlated with the subharmonic amplitude. The results show that albumin-stabilized microbubbles, similar to those currently used as US contrast agents, may provide a significant source of nuclei and improve prospects for cancer therapy using acoustic cavitation. The evidence presented supports the hypothesis that cell damage is due to a sonochemical rather than to a sonomechanical process.

Sonochemical activation of hematoporphyrin: an ESR study.

The production of 2,2,6,6-tetramethyl-4-piperidone-N-oxyl by reaction of 2,2,6,6-tetramethyl-4-piperidone (TMPone) with ultrasonically generated active species in oxygenated solutions of hematoporphyrin (Hp) was studied by electron spin resonance spectroscopy. The nitroxide production rate in air-saturated TMPone solutions in phosphate-buffered saline of pH 9.0 was significantly higher in the presence of Hp than in its absence. The enhancement of nitroxide production by Hp was significantly inhibited in the presence of sodium azide or histidine in the solution. The production rate with Hp was doubled by substitution of deuterium oxide, while the rate without Hp increased only modestly. These results suggest that a substantial amount of active oxygen can be generated by ultrasound in aqueous solutions of Hp. Since the production rate was not reduced by mannitol and no nitroxide was produced in nitrogen-saturated solutions, it appears that hydroxyl radicals do not account for a major portion of the active oxygen species which reacted with TMPone to yield a nitroxide.

Mode scanning: heating pattern synthesis with ultrasound phased arrays.

Modes, the characteristic symmetric focal patterns of an ultrasound phased array, prove especially useful for hyperthermia. Modes cancel the complex pressure fields exactly along the central axis, eliminating axial constructive interference both proximal and distal to the treatment volume. A simple calculation exploits planar or rotational array symmetry and produces the driving signals which generate modal focal patterns. Results show that temporal mode scanning improves heating patterns considerably, expanding the maximum treatable size of the tumour volume.

Insonation of fixed porcine kidney by a prototype sector-vortex-phased array applicator.

The sector-vortex applicator, an ultrasound phased array with a geometric focus having multiple sectors and tracks, can directly synthesize, without scanning, diffuse focal patterns useful for hyperthermia. A perfused tissue phantom, consisting of an alcohol-fixed porcine kidney with thermocouples placed in the cortex, is insonated by a prototype sector-vortex applicator with 16 sectors and two tracks at an ultrasound frequency of 750 kHz. Steady-state temperature distributions are measured for a wide range of perfusion rates. Results demonstrate that the radius of the heated region can be controlled effectively by choosing the focal mode of the applicator as it is predicted by theoretical analysis.

Direct computation of ultrasound phased-array driving signals from a specified temperature distribution for hyperthermia.

This paper presents a new method which obtains ultrasound hyperthermia applicator phased-array element driving signals from a desired temperature distribution. The approach combines a technique which computes array element driving signals from focal point locations and intensities with a new technique which calculates focal point locations and power deposition values from temperature requirements. Temperature specifications appear here as upper and lower bounds within the tumor volume, and a focal point placement algorithm chooses focal patterns capable of achieving the temperature range objective. The linear algebraic structure of the method allows rapid calculation of both the phased-array driving signals and an approximate temperature field response. Computer simulations verify the method with a spherical section array (SSA) for a variety of temperature specifications and blood perfusion values. This scheme, which applies to any phased-array geometry, completes an essential step in both treatment planning and feedback for hyperthermia with ultrasound phased-array applicators.

Radiant heat-induced hyperthermia in mice: in vivo effects on the immune system.

Whole-body hyperthermia (WBH) in mice was induced by 2-4-h exposure to radiant heat resulting in core body temperatures of 38.5-40.4 degrees C, and correlated directly with the magnitude and duration of heat treatment. Two-hour heat treatments in this temperature range did not consistently affect generation of antibody-forming cells in vivo, while 4-h treatments at temperatures greater than or equal to 40 degrees C significantly suppressed the antibody-forming cell response. The capacity of lymphocytes from similarly heated mice to generate antibody-forming cells in vitro was not affected, suggesting that the observed in vivo suppression may be mediated by circulating factors rather than by some heat-induced alteration in the cells themselves. In vivo treatment did not alter T-cell responsiveness to the mitogen Con-A or delayed-type hypersensitivity responses to sheep red blood cells. Quantitative and flow cytometric assessment of splenic and thymic lymphocyte numbers showed that WBH did not alter absolute numbers of lymphocytes but did temporarily change the proportions of lymphocyte subsets. An immediate increase in splenic L3T4+ cells was observed, followed within 18 h by an overall decrease in Lyt 2+ and Thy 1.2+ T-cells. In the thymus the percentages of mature T cells increased. In general, only minimal effects of heat on the immune responses of normal mice could be demonstrated.

Acoustical evaluation of a prototype sector-vortex phased-array applicator.

A prototype sector-vortex phased-array applicator for ultrasound hyperthermia was constructed and acoustically evaluated. The array transducer consists of special lead-titanate ceramic elements of 16 sectors and two tracks attached on a element is driven by a complementary pair of power MOSFETs at 750 kHz. An annular focal field approximated by the Mth order Bessel function is theoretically predicted to be formed when the array elements are driven with a phase distribution that rotates M (

Optimization of the intensity gain of multiple-focus phased-array heating patterns.

A new technique for enhancing the intensity gain at the focal points in multiple-focus patterns is introduced. The new technique is shown to be effective in reducing the interference typically associated with multiple-focus patterns. This reduction in interference patterns allows multiple-focus scanning to generate highly localized heating. Simulation results indicate that multiple-focus scanning not only provides an alternative to single-focus scanning, but also achieves better localization in the heating pattern. The maximization of intensity gain of multiple-focus heating patterns significantly reduces the pre-focal-depth high-temperature regions that can be caused by single-focus scanning. This is shown by computer simulation of a two-dimensional cylindrical-section array (CSA2D) as a heating applicator. Two series of simulations are presented in which different scan trajectories were used to therapeutically heat a small deep-seated target volume. In every case the heating pattern was generated using single-focus scanning and multiple-focus scanning (with and without intensity gain maximization). Multiple-focus scanning with gain maximization offers the best localization of heating to the target volume of the three methods.

A spherical-section ultrasound phased array applicator for deep localized hyperthermia.

Computer simulation shows that a new ultrasound phased-array with nonplanar geometry has considerable potential as an applicator for deep localized hyperthermia. The array provides precise control over the heating pattern in three dimensions. The array elements form a rectangular lattice on a section of a sphere. Therefore, the array has a natural focus at its geometric center when all its elements are driven in phase. When compared to a planar array with similar dimensions, the spherical-section array provides higher focal intensity gain which is useful for deep penetration and heat localization. Furthermore, the relative grating-lobe level (with respect to the focus) is lower for scanned foci synthesized with this array (compared to a planar array with equal center-to-center spacing and number of elements). This could be the key to the realization of phased-array applicator systems with a realistic number of elements. The spherical-section array is simulated as a spot-scanning applicator and, using the pseudo-inverse pattern synthesis method, to directly synthesize heating patterns overlaying the tumor geometry. A combination of the above two methods can be used to achieve the desired heating pattern in the rapidly varying tumor environment.

Effect of phase errors on field patterns generated by an ultrasound phased-array hyperthermia applicator.

The effect of phase quantization errors and Gaussian distributed random phase errors on field patterns synthesized by a rectangular ultrasound phased array hyperthermia applicator is studied. The parameters defined show that, over the range of four-bit to one-bit quantization, the simulated, patterns degrade with increasing phase errors. However, the overall shape and position of the foci remain unchanged. Simulation results show that, even with one-bit phase quantization, or with a Gaussian distributed random phase error of standard deviation of about 52 degrees , the applicator can still produce useful, if not particularly clean, power deposition patterns. Thus, the power deposition patterns are remarkably stable tolerating quite large phase error levels. This suggests that the power deposition patterns inside the body may be relatively insensitive to phase errors due to tissue inhomogeneities.