Ultra-rare-event detection performance of a custom scanning cytometer on a model preparation of fetal nRBCs.

BACKGROUND: The performance of a fully automated scanning cytometer incorporating previously reported high-precision autofocus and accurate image segmentation was evaluated for the detection of "ultra-rare" cells using a model of fetal nucleated red blood cells (fnRBCs) in the maternal circulation. These distinctive scanning cytometry techniques were expected to markedly improve sensitivity and specificity. METHODS: Normal adult blood and fetal red blood cells were stained with fluorescein isothiocyanate-conjugated anti-fetal hemoglobin and 4,6-diamidino-2-phenylindole, a nuclear dye. Adult cells were spiked with fetal cells to create ratios of about 1 fnRBC in 10(7) nucleated cells and deposited in monolayers on slides using centrifugal cytology. Rare-event performance parameters were reviewed, formalized, and applied to test the new instrument using this cell model. RESULTS: Fifteen slides were analyzed to establish performance by comparison with manual detection, and four sets of four slides each were then scanned to explore the limit of detection. Results were an average sensitivity of 91%, an average specificity error of 12.3 false-positives per million cells, and repeatability of 100% at a cell analysis rate of 862 Hz. With addition of a quick interactive step subsequent to scanning, the false-positive rate dropped to a total of only one artifact over the 15 experiments. The instrument succeeded at locating 1 fnRBC in 20 million adult cells, the lowest limit of detection tested. CONCLUSION: This consistently high performance, coupled with the capability of scanning arbitrarily large numbers of cells, validates the considerable potential of precise high-speed autofocus and accurate real-time image segmentation for ultra-rare event detection.

Use of a spherical multiparameter transducer for flow cytometry.

Fused silicon dioxide, multiparameter flow transducers with 50 microns internal square cross section and approximately 60 microns length can simultaneously measure DC and RF impedance as well as fluorescence and multiple-angle light scattering. A spherical version of such a transducer was mounted in an EPICS CVA flow-cell housing and was installed on a research prototype equipped with an argon-ion laser. The signal that was produced by the spherical transducer with EPICS DNA-Check beads was 1.73 times greater than that produced with the standard cylindrical flow cell. Similarly, with EPICS Immuno-Brite beads, the average ratio was 1.96. The Coulter impedance and light-scattering measurements were similar to those produced with the conventional cylindrical outside flow cell, although the internal cross section of the sphere was square and that of the cylinder was circular. The theoretical arguments of Leif and Wells have been demonstrated to be correct. At present, monolithic, spherical fused-silica transducers are the optimal design for combined electrooptical, multiparameter flow cytometry analyzers.

Ada software for cytometry.

Ada is a new general-purpose language that embodies the concepts of software engineering. Although it was initially developed for military purposes, it is suitable for developing software for cytometry and other health-related applications. A pilot study has demonstrated the feasibility of employing Ada for cytometry applications. Three packages were created. The first subtracts a control three-dimensional population from multiple individual experimental populations and presents the results in spread sheet form. A second package has the capability of finding aggregates of cells. The results of this package are visualized employing a commercially available program for three-dimensional presentation of the data that permits rotation in real time. A third package consists primarily of interface drivers for two commercially available personal computer boards, an ADC and a stepper motor controller. The major problems with the coding were due to incomplete implementation of the language. This pilot study, together with others, indicates that it would be both cost effective and beneficial to implement cytometry and other medical devices in Ada.

The effects of tissue-associated and MHC class II antigen presentation on in vitro lymphoproliferative responses against canine liver and kidney cell subpopulations.

Purified hepatocytes (LH), Kupffer cells (LKu), and intrahepatic biliary duct cells (LD) were isolated from canine livers, as well as tubular cells from canine kidneys, by enzymatic digestion, gradient centrifugation, and tissue culture techniques. Incubation of LH, LKu, and LD for 48 hr in a two-compartment diffusion chamber opposite two-way mixed lymphocyte cultures, or with canine gamma interferon purified and standardized in our laboratory, resulted in a significant increase in class II expression. This was detected in the cell analyzer with directly fluoresceinated B1F6, a monoclonal antibody (mab) generated in our laboratory vs. a canine class II monomorphic epitope. An amplification of the allogeneic mixed lymphocyte liver cell cultures (MLLC) of at least 2-fold was observed by preinduction of canine class II expression with IFN-gamma on LKu and LD cells, but an autologous reaction could not be elicited. However, an autologous as well as allogeneic lymphoproliferation against kidney tubular cells (MLKC) could be easily observed without IFN-gamma and amplified with IFN-gamma to stimulation indices of at least 3 times that of noninduced cultures. Dependence of the allogeneic MLLC and allogeneic and autologous MLKC on class II gene expression was also evidenced by blocking of 3H-thymidine uptake seen by incubation with 5 micrograms of B1F6. Another mab, I1F6, generated against tubular cells and inhibiting the autologous and allogeneic MLKC, had no blocking effect on lymphoproliferation with any of the liver cell preparations. No such tissue-specific mab (analogous to I1F6) has thus far been found in response to mouse immunization with LH, LKu, or LD. In the absence of accepted defined molecular probes in the dog as yet, we conclude that, in contrast to kidney tubular cells, cells of the normal canine liver do not readily stimulate a primary lymphoproliferative autoimmune reaction in vitro despite class II amplification. Thus autoreactivity (as opposed to alloreactivity) is much less prominent in immune recognition of purified cellular components of nondiseased liver tissue than of kidney tissue in which tissue-associated epitopes are more operative.

Electrophoretic mobility studies on doxorubicin-resistant and -sensitive murine P388 leukemic cells.

The electrokinetic properties of doxorubicin (DOX)-resistant (P388/R) and -sensitive (P388/S) murine leukemic cells were studied in a free-flow electrophoresis (FFE) system. The electrophoretic mobilities (EM) of P388/S and P388/R cells were 1.07 and 1.35 x 10(-4) cm2 V-1 s-1, respectively, suggesting a higher net negative charge on the P388/R cells. Neuraminidase treatment decreased the EM of both the P388/S and P388/R cells by 15-20% but had no effect on cellular doxorubicin retention. Total and cell surface sialic acid contents were similar in both the cell lines. Our studies show that no direct correlations may exist among surface charge, cell surface sialic acid content, and doxorubicin retention in DOX-resistant and -sensitive P388 cells; however, differences in cell surface charge between these cell types were used to separate them by preparative FFE.

Optical analysis of the AMAC IIIS transducer.

The AMAC IIIS electrooptical transducer for flow cytometry facilitates simultaneous measurements of electrical impedance and several optical properties of single cells. Ray-trace analysis demonstrates that its spherical exterior permits light rays to emerge effectively undeviated and thus lowers the required numerical aperture of the collecting lens as compared with a flat surfaced transducer. This feature results in an increased light-gathering ability, an increased depth of focus, minimum spherical and chromatic aberrations, and minimizes the optical volume observed.

The EPICS C analyzer. An ergometrically designed flow cytometer computer system.

A description is presented of a new flow-cytometer (FCM) computer system, the EPICS C analyzer, which was developed to be used by a moderately trained technician as opposed to an FCM expert. The system was made robust and user-friendly by the use of menu-based rather than command-driven software. The user primarily interacts with the self-explanatory menus by depressing one of the eight membrane selector switches located on each side of the screen. The minimizing of typing and restriction of the user's responses greatly simplify the use and design of the software. The EPICS C operates in two modes: as a reliable clinical instrument and as a research tool. The instrument settings are stored on disk files rather than manually logged into a book. The settings for the predefined tests are read from the disk.

Two-dimensional impedance studies of BSA buoyant density separated human erythrocytes.

Combined DC (Coulter Volume) and radio frequency impedance studies were performed on human erythrocytes which had been separated by buoyant density in linear, neutral, isotonic bovine serum albumin gradients. The individual buoyant density fractions showed no reproducible shift in volume with buoyant density but did show a shift with opacity, radio frequency impedance divided by dc impedance. This new electronic parameter of opacity can be related to cell age, since both it and cell age are directly related to buoyant density. This increase in opacity with buoyant density is correlated with a change in shape.

Convention on nomenclature for DNA cytometry. Committee on Nomenclature, Society for Analytical Cytology.

Analysis of cellular DNA content by cytometry is important in clinical and biological research. Measurements are used widely to assess the relative DNA content of tumor stemlines and to assist in the detection and evaluation of malignant diseases. A review of the literature on DNA measurements in solid tumor and leukemias reveals a confusing variety of terms applied for the description of similar results. In order to facilitate the understanding of data and to standardize the terminology for DNA analyses, a questionnaire was distributed to more than 500 investigators. Subsequently, a workshop on terminology was held at the Combined Conference on Analytical Cytology and Cytometry IX and VIth International Symposium on Flow Cytometry, Schloss Elmau, West Germany, 18-23 October, 1982. The workshop nominated a nine-member committee to develop guidelines for nomenclature to be used in reporting results from analyses by DNA cytometry. The committee was charged by the Council of the Society for Analytical Cytology to complete this task and to publish its recommendations in Cytometry and in Cancer Genetics and Cytogenetics. The following guidelines are based on the questionnaires returned and the discussion at the workshop; they represent the unanimous recommendations of the committee. The five guidelines given herein apply to measurements of relative DNA content of cells that have been stained appropriately and analyzed by cytometry.

Centrifugal cytology of ocular fluids.

Centrifugal cytology is a new technique for the preparation of ocular fluids for cytologic examination. It differs from conventional cytocentrifuge techniques, since fixation is carried out simultaneously with centrifugation. It avoids air-drying artifacts and can be applied to small (50 microL) or large (200 mL or more) sample volumes and dilute cell suspensions, such as might be obtained by anterior chamber paracentesis, vitreous aspiration, or vitrectomy. The resultant preparation is a permanent, stained, well-preserved, flattened, homogeneous cell dispersion suitable for detailed cytologic evaluation.

An immunofluorescence method for monitoring DNA synthesis by flow cytometry.

An immunofluorescence method for monitoring DNA synthesis in single cells has been developed for flow cytometry. With antiserum which is specific for 5-bromodeoxyuridine (BrdUrd) and a second fluorescent label, BrdUrd-incorporation pulses of 30 min are detectable. The fluorescence intensity of the incorporated BrdUrd, as determined by immunofluorescence, is related to the amount of BrdUrd incorporated, as shown by isotopic methods and cell sorting. Thus, the technique may be applicable to determining rates of replication per cell. Multiple samples of as few as 1 X 10(5) cells can be fixed, hydrolyzed and treated with the anti-BrdUrd antiserum. Nuclear-bound IgG is localized by fluorescein-labeled avidin-D. Since the technique uses whole cells, other parameters such as light scatter and DNA content can be simultaneously monitored so that cohorts of "labeled" cells can be followed through the cell cycle.

Cervical carcinoma antigens in the diagnosis of human squamous cell carcinoma of the cervix.

The use of tumor-associated antigens (TAA) in the diagnosis of human squamous cell carcinoma of the cervix was evaluated by immunofluorescence staining of second cervical scrapes and touch preparations of normal and carcinomatous tissue. Rabbit antisera, prepared against human cervical squamous cell carcinoma homogenates and absorbed with normal human cervix and plasma, were used to stain 103 second cervical scrapes by indirect immunofluorescence. Of these specimens, 59 were positive by immunofluorescence, whereas the remaining 44 were negative. Compared with conventional cytologic diagnosis, positive immunofluorescence was detected in 100% (49/49) of the second scrapes from patients with cervical dysplasia or carcinoma (for a false-negative rate of zero). Of the second cervical scrapes from 57 patients negative by cytology, 13 were positive by immunofluorescence (for a false-positive rate of 22.8%). Indirect immunofluorescence tests on tumor touch preparations also revealed cervical TAA in other types of gynecologic tumors.

Centrifugal cytology of nipple aspirate cells.

Two improvements in the methodology for obtaining and preparing nipple aspirates from nonlactating women are reported. The first is the development and use of a new breast pump with a controllable vacuum and cups of various sizes. The second is the use of centrifugal cytology to prepare the dispersions. Twenty-one of 24 breasts of patients in the age range 30 to 49 years produced cellular dispersions which contained foam cells; of them, 13 contained ductal cells. A comparison of glutaraldehyde and ethanol fixation indicated that the cells appeared substantially the same.

The dissociation and separation of bovine adenohypophysial cells.

Bovine adenhypophysial tissue was dissociated by sequential enzymatic incubation in a continuous flow system. Dispersed cells separated into discrete fractions after centrifugation in isopycnic bovine serum albumin gradients. The dispersed and separated cells were prepared for microscopic identification and differential counts by centrifugal cytology. Radioimmunoassays for LH, FSH, TSH, and Prl were used to corroborate the differential counts and determine the homogeneity of the fractions. The thyrotrophs banded at an average density (rho) of 1.0417, the FSH-secretory cells at rho = 1.0597, the LH-secretory cells at rho = 1.0458, and the Prl-secretory cells at rho = 1.0126. A 7-16 fold enrichment of different cell populations was possible. In bovine hypophyses each hormone appears to be formed by specific cells: the average TSH concentrations of the thyrotrophs were 5.1 pg/cell for LH- and FSH concentration were 4.7 and 4.9 pg/cell for LH- and FSH-secreting cells, respectively. The average Prl concentration was 4.9 pg/cell for Prl-secreting cells.

The automated multiparameter analyzer for cells (AMAC) IIA, a true bridge circuit Coulter-type electronic cell volume transducer.

A true bridge Coulter effect (electronic cell volume) transducer has been developed. All resistances of this bridge are now the result of current flow through saline channels. Contamination by electrode products including gas bubbles has been completely eliminated since both power electrodes are now remote from the flow chamber. Since the orifice is in series with an approximately 10 K ohm resistance generated by a gel-filled capillary and a displacement rheostat, it floats electrically, at virtual ground. The other side of the bridge consists of a fluid side-wire. Removing the power electrode from the orifice outlet makes possible downward flow and the use of a single outer sheath, and eliminates noise generated by gas bubbles which could possibly be trapped. It should now be possible to combine this design with that of the AMAC III square orifice, to produce an electro-optical sorter where all parameters are measured simultaneously. This true bridge circuit possesses the further advantage that noise due both to the power supply and to overvoltage at the power electrodes is common-mode rejected, and any drift due to changes in electrode polarization is eliminated. Preliminary experiments confirm results with the AMAC II that hemoglobinopathies can be recognized by the increased coefficient of variation (CV) of the erythrocyte spectra.

A permanent cell viability assay using alcian blue.

The alcian blue dye exclusion method for glutaraldehyde-fixed cells has been utilized with "centrifugal cytology" to prepare permanent records of the viability of individual cells present in suspensions. The viability of spleen cell suspensions separated by linear bovine serum albumin density gradient centrifugation has been measured with this method. Combined light and scanning electron microscopy of nonviable and viable cells demonstrated membrane alterations in alcian blue-stained nonviable cells, while viable cells were spherical and displayed uniform surface features.

Development of instrumentation and fluorochromes for automated multiparameter analysis of cells.

We have developed and interfaced to a computer an automated instrument (the AMAC III) which is designed to observe simultaneously several physical parameters of cells. Typical parameters include electronic cell volume (Coulter effect), RF amplitude (opacity), multiwavelength fluorescence of cytological stains, and cell light-scattering. The use of a new ultraviolet laser combined with a holographic grating spectrograph promises to increase the number of fluorescing species that can be detected simultaneously. This number can be further increased by use of special rare-earth-based fluorochromes, that emit well-defined, spectrally distinct peaks.