Hum. Genet. 47, 319--327 (1979) © by Springer-Verlag 1979

An Automatic System for Chromosomal Analysis Applied to Prenatal Diagnosis C. L60nard 1, P. Saint-Jean 2, D. SchoEvaErt 1, P. Eydoux 3, S. Girard 4, and R. Le G62. Laboratoire d'Histologie-Embryologie-Cytog6n6tique, C.H.U. Bic~tre, F-94270 Kremlin-Bic~tre, France 2Commissariat fi l'Energie Atomique, Institut de la Protection et S6curit6 Nucl6aire, DPR-SRP, B.P. 6, F-92260 Fontenay-aux-Roses, France 3Laboratoire d'Histologie-Embryologie-Cytog6n~tique, C.H.I., F-78303 Poissy, France 4Laboratoire d'Anatomo-Pathologie-Cytog6n~tique, C.H. Saint-Vincent-de-Paul, 74, avenue Denfert Rochereau, F-75014 Paris, France

Summary. Computer-assisted metaphase analysis for prenatal diagnosis can be economically performed by a preexisting hardware-software system. The program can be run by technician-level (i.e., nonspecialist) personnel who can opt for the automatic operational mode for good quality metaphases or for the semimanual mode for problem metaphases (numerous superpositions, artifacts, etc.). The karyotype can be obtained in conversational mode. Hardcopy output is available for all modes.

The increasing demand for chromosomal analysis in various medical specialties has led, in several countries, to the development and perfection of a number of information systems to render automatic and rapid certain time-consuming and tedious stages of chromosomal analysis (Butler et al., 1964; Ledley et al., 1966; Mendelsohn et al., 1969). The purpose of this study was to determine the level of aid provided by one of the existent systems at the CEA 1 with the aim of processing chromosomal analyses for prenatal diagnosis. This preliminary study was especially intended for one of the major reasons for prenatal diagnosis, the screening for anomalies of chromosomal number that occur more frequently with increasing maternal age. The CEA system was essentially designed for chromosomal analysis of blood lymphocytes. The problem is that lymphocytic metap h a s e s - - w h o s e chromosomes are short and well separated--differ morphologically from the metaphases of fetal cells, which have long slender chromosomes and numerous superpositions. Thus, one of the aspects of this study was to ameliorate chromosomal-analysis processing, with respect to both the processing system for digitizing the images and the methods of biologic preparation of the material, to optimize its applica* To whom offprint requests should be sent 1 French Atomic Energy Commission. Radiopathology Service of Dr. R. Le G6

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tion to fetal chromosomal a n a l y s e s . T h e s e m o d i f i c a t i o n s will f a c i l i t a t e t h e digitization of the images, accelerate chromosomal enumeration, and render their i d e n t i f i c a t i o n m o r e c o n v e n i e n t f o r t h e o p e r a t o r , p a r t i c u l a r l y t h e o p e r a t o r w h o is not a specialist in information processing.

Chromosome Preparation The culture technique has been described by Bou6 and Bou6 (1973) and Bou6 et al. (1976): the amniotic fluid is cultured, without previous centrifugation, in eight to ten petri dishes of 35-mm diameter, containing a circular coverslip that completely covers the bottom. The dishes are then filled with 1.5 ml of amniotic fluid and 1.5 ml of RPMI 1640 supplemented with 20% fetal calf serum. The material is then placed at 37°C in a CO2 incubator. From the fourth day, culture medium is changed every two days and cellular growth is monitored with an inverted microscope. When cell colonies containing numerous mitoses are observed, chromosome preparations can be obtained (between days 8 and 12): 20h after the culture medium is changed, the slide is immersed in a hypotonic solution (Hanks solution 9: 1), for 15min at 37°C. The preparation is then fixed (ethanol-acetic acid 3 : 1) at 20°C and air-dried. The slide can then be stained using any chromosome technique. For our study we used either Giemsa or the trypsin denaturation technique of Seabright (1971): denaturation in 0.25% trypsin solution from 30 to 90 s at 20 ° C, PBS rinse, and 10 min of 4% Giemsa stain, buffered at pH 6.8. Stained cell colonies are easily located by the naked eye. The metaphases have also been located using a 100x magnification (this selection can also be performed on an automatic microscope mitosis-selector system of the EIDOMAT type) (Le G6, 1969). The analysis was performed using transmitted light with a green filter and an oil-immersion objective 63x.

The Analysis System The system includes (1) an image captor coupled to a preanalysis device, the ASTI, (2) a minicomputer pattern recognition and automatic classification, and (3) a software system, as described in previous papers (Le G6, 1969, 1972; Le G6 et al., 1977; Spiwack, 1976).

The Image Captor (ASTI) This system was constructed by the Electronic Service of Saclay (SES-CEA) in collaboration with the Radiopathological Service under the direction of Dr. Le G6. The system functions in two stages (Fig. 1):

1. Digitization. A Thomson standard TV camera is connected to the microscope. The video signal is digitized by a numerical analog computer (C.A.N.) with 16 gray levels. Among these, a single gray level is defined as one being useful for differentiating the image from the background. Numerical information is condensed by compression of the data above threshold and stored in a 32K octet MOS memory. The image definition is then at 400 points per line on 2 x 286 interdigitating lines. The image thus obtained is visualized after decompression and analog numeric conversion (C.N.A.) on a standard television screen to control the on-line quality of data acquisition. At this point the operator can regulate image clarity, centering, and contrast. Once the image has been acquired, the data stored in memory are permanently displayed and the microscope is then freed from the input loop. (An operator can then proceed with image analysis while another can use the microscope to locate another metaphase. The coordinates of the previous metaphase are noted so that it can be located immediately if verification is necessary.) 2. Preanalysis by the ASTI (Automate Sp6cialis6 de Traitement d'Images): There are three possible modes of image analysis that can be used directly in the MOS memory by cabled logic under control of the operator: command console, joy stick, and a vector on the television screen.

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First, the global automatic analysis: All isolated elements composing the image are counted (isolated chromosomes or groups of chromosomes or artifacts). Only a perfectly spread mitosis on a clean background can give an exact count of the image ensemble. Secondly, the sequential automatic analysis: Each element is sequentially selected in scanning order by using a step-by-step command activated by the operator. This element is visualized separately, and its parameters are displayed on the command display unit: the area, the weighted gray-level area, and the screen coordinates. Thirdly, the manual analysis: The operator can perform four functions: (1) select an object by the vector visualized on the screen and commanded by the joy stick; (2)isolate the selected object (display of the parameters); (3) mask the object selected by the vector (the element no longer enters into the automatic analysis: elimination of artifacts); (4) section the interior of a designated object by creating a frontier, i.e., a broken line constructed by using a vector that can be regulated in length, direction, and position (separation of superposed chromosomes). Using these three modes of analysis, it is possible to furnish the computer with the matrix of points that defines a chromosome (vignette) and only that chromosome. For each chromosome, the operation is repeated. The Information System This comprises (1) a multi 20 central memory (Intertechnique) with a 32 K octet memory, (2) a 2.5 megaoctet removable cartouche disc, and (3) a Tektronix 4015 graphic console with hardcopy output.

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Fig. 2. a Analysis of a good quality metaphase (46,XY). b (Hardcopy) Reproduction of a chromosomal analysis (execution time 20 s, 44 elements enumerated). The image contains touching and superposed chromosomes (elements 04 and 31) and a noisy nuclear artifact (element I4). e Automatic hardcopy after masking the ambiguous elements (20 s for 41 elements). The manual interventions involved 3 erasures, d Final document (30 s for 46 elements). Addition to Figure 2c of the chromosomes separated by manual cuts. The operations illustrated here required about 2 min

The Software System This system includes a large number of functions designed for the morphologic analysis of chromosomes and their identification. These pattern-recognition functions are activated sequentially by a monitor, which has been designed with a system of overlays.

Method and Results of Analysis at a Constant Magnification The sequence o f analyses necessary to o b t a i n the c o m p l e t e c h a r a c t e r i z a t i o n o f a m e t a p h a s e includes the following:

In Case of a Good Quality Metaphase (Fig. 2): The o p e r a t o r , having o b t a i n e d the mitosis b y A S T I , m a s k s the artifacts t h a t at first resemble c h r o m o s o m e s (similar sizes) a n d the c h r o m o s o m e s u p e r p o s i t i o n s that can occur in small n u m b e r s . The software then effects the transfer o f the vignettes so o b t a i n e d sequentionally, e l i m i n a t i n g a u t o m a t i c a l l y those, small o r large, typical o f dust particles o r cellular debris (nuclei, c y t o p l a s m i c remains). F o r each vignette r e t a i n e d the p r o g r a m calculates the c o n t o u r a n d the h o m o t h e t i c p o s i t i o n on the g r a p h i c

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Fig. 3a--d. Analysis of a metaphase (47,XY,21+) with numerous superpositions (Fig. 3a): Steps 0a), (c), and (d) occurred in the same sequence as in Figure 2 with: 20s and 37 elements for (b), 20s and 30 elements for (c), 8 rain and 47 elements for (d) (11 isolations). It requires about 10min to analyze this type of mitosis

screen on the basis o f the coordinates o f the vignette frame. It then traces the c h r o m o s o m e on the screen with its acquisition order number. The o p e r a t o r works in conversational m o d e with the software to transfer one by one the remaining c h r o m o s o m e s that had been cut out using the mobile vector. C h r o m o s o m e s that could not be initially disassociated are then graphically displayed; they still appear superposed, but each is individually outlined and assigned a distinct identification number. We thus obtain a facsimile o f the mitosis with its enumeration on the graphic display unit.

In Case of a Poor Quality Metaphase (numerous superpositions, dispersed metaphases, clumped metaphases) (Fig. 3): The conversational m o d e is necessary for the acquisition of succeeding c h r o m o s o m e s once they have been cut out by the vector. Widely dispersed metaphases must be processed in two phases if the moveable microscope stage cannot be positioned to include all o f the field. But the resulting k a r y o t y p e will be positioned as a whole on the h a r d c o p y with its coherent enumeration. Once the c h r o m o s o m e s have been input and analyzed, after a final visual verification o f the c o n c o r d a n c e o f the microscopic and display screen images, they can be o u t p u t at will either on h a r d c o p y o f the facsimile (i.e., the h a r d c o p y of the viewing screen with the set o f c h r o m o s o m e s in their initial position, outlined and numbered) (Fig. 2d and Fig. 3d) or as a conventional karyotype estab-

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lished by conversational mode, or semiautomatically, as was the case for the lymphocyte metaphases. The instrumentation and software just described are able tO produce an exact enumeration of the chromosomes of fetal cell metaphases. This analysis has been possible with the existing system for all of the mitoses presented (about 20). But given the nature of the fetal preparations such as closely aligned chromosomes and complex superpositions, which are common, the operator has had to resort frequently to the conversational mode. For an experienced operator analyzing a well-spaced metaphase containing a few simple superpositions (Fig. 2), the total time required for the final hardcopy document is at present 5-10 min. In case of difficult metaphases among those usually selected (clumped, multiple, and complex superpositions, and artifacts) (Fig. 3), the analysis requires 20-30rain. A biologist not specialized in information processing can rapidly learn to manipulate the system (after about 20 analyses).

Discussion

The automatic analysis of metaphases of fetal cells grown from amniotic fluid is more demanding than that of blood lymphocytes because of the fineness of the chromosomes, which are rarely straight and very often superposed. In the enumeration step, the metaphase clumping factor has the unfortunate consequence of artificially uniting two chromosomes that are close into a convex image, and thus necessitating the conversational mode to obtain a correct separation. Therefore, for this type of analysis it is extremely important to find a biologic method, such as producing maximum hypotonic treatment in order to separate chromosomes as much as possible from one another. The usual problems of adjusting such an acquisition system is related to the homogeneous lighting of the field to be analyzed: the operator must consider two usual factors to achieve proper adjustment. First, a cytoplasmic background that is denser in the center than in the periphery. Then, the unequal distribution of the light transmitted to the microscopic field, which is related to the presence of the bulb filament: the illuminated background as seen by the camera is lighter in the center than at the periphery. These two phenomena partially compensate each other. The operator must, by displacing the metaphase and by using the optical system of the microscope, find on the control display the greatest luminous uniformity of the field to be analyzed, such as it is to be memorized. The resolution (400 points per 286 lines) obtained on a television screen of 40 cm on the diagonal produces an image that is not as well focused as that of the direct image. But efforts to obtain better resolution run into problems caused by the appearance of noisy discontinuities in the light bands of denatured chromosomes with densities too similar to the background. These problems do not occur in the present system. The analysis time reported here can be shortened by removing several timeconsuming factors. Although the automatic analysis is very rapid, the necessity of frequently using the vector to separate and isolate the chromosomes represents the most time-consuming part of the analysis. It has led to the replacement of the

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v e c t o r b y a light pen, which p e r m i t s the o p e r a t o r to outline the i m a g e in a curvilinear a n d c o n t i n u o u s fashion. This system is presently being tested on a new console specialized in c o n v e r s a t i o n a l mode. Lastly, the c h r o m o s o m e s o f fetalcell m e t a p h a s e s have c h r o m a t i d s t h a t lie very close t o g e t h e r a n d require a p r o g r a m b e t t e r a d a p t e d to this p r o b l e m . This s t u d y has s h o w n t h a t a system presently exists that, with some i m p r o v e ments, c o u l d replace the m o s t t i m e - c o n s u m i n g a n d tedious aspects o f p r e n a t a l diagnosis, m e t a p h a s e analyses. This system m a k e s it possible to d i s p l a y visual optical m i c r o s c o p i c fields on a c a t h o d e display, to isolate a u t o m a t i c a l l y or m a n u a l l y each c h r o m o s o m e , to p r o d u c e a h a r d c o p y facsimile o f the c h r o m o s o m e display, a n d to establish a k a r y o t y p e in the c o n v e r s a t i o n a l m o d e a n d o b t a i n a h a r d c o p y facsimile o f it.

Acknowledgements. The authors thank Pr. A. Bou6 for his helpful suggestions and criticisms and Victoria von Hagen for help in the preparation of the manuscript. This study was supported in part by grants from the INSERM (ATP 36/76/68) and from the Delegation for Protection and Nuclear Security of C.E.A.

References

Bou6, J., Bou6, A.: L'int6r& en diagnostic pr6natal des techniques nouvelles d'identification chrom0somique dans des translocations et une aneusomie de recombinaison. Nouv. Presse Med. 2, 3097--3102 (1973) Bou6, J., Bou6, A., Girard, S., Thepot, F.: Diagnostic pr6natal des anomalies chromosomiques. R6sultat de trois ann6es. Arch. Fr. Pediatr. 33, 653--664 (1976) Butler, J. N., Butler, M. K., Strond, A.: Automatic classification of chromosomes. Oxford Pergamon Press 3, 261--275 (1964) Ledley, R. S., Rotolo, R. S., Wilson, J. B., Belson, M., Golab, T. J., Jacobson, J.: Pattern recognition studies in biomedical sciences. Proc. Spring Joint Computer Conference. Boston (Mass.), pp. 411--430 (1966) Le G6, R.: L'analyse chromosomique par reconnaissance automatique des formes. Colloque international sur la reconnaissance des formes. Grenoble, 11--13 Sept. 1968, CEA. LETI, pp. 399--410, 1969 Le G6, R.: Etudes pour une m6thode d'automatisation des analyses chromosomiques. Thbse de l'Universit6 Paris VI UER 65, Rapport CEA R. 4325, 1972 Le G6, R., Saint-Jean, P., Khochtinat, N., De Cosnac, B.: Automatisation du caryotype au moyen d'un appareillage sp6cialis6 de traitement d'images. IRPA, 4, 1293--1296 (1977) Mendelsohn, M. L., Hungerford, D. A., Mayall, B. H., Perry, B., Conway, T. J., Prenitt, J. M. S.: Computer-oriented analysis of human chromosomes. Ann. N.Y. Acad. Sci. 157, 376--392 (1969) Seabright, M.: A rapid banding technique for human chromosomes. Lancet 1971 II, 971 Spiwack, A.: Automate sp6cialis6 de traitement d'images (ASTI). Th~se de l'Universit6 Paris Sud, Orsay, 1976 Received September 9, 1978