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BANDSCAN — A computer program for on-line linear scanning of human banded chromosomes
Computer Programs in Biomedicine 8 (1978) 283-294 © Elsevier tNorth-Holland Biomedical Press
B A N D S C A N - A COMPUTER PROGRAM FOR ON-LINE LINEAR SCANNING OF HUMAN B A N D E D CHROMOSOMES Mfiximo E. DRETS Division of Human Cytogenetics, Instituto de lnvestigaciones Biol6gicas Clernente Estable, Avda. Italia 3318, Montevideo, Uruguay BANDSCAN, an interactive program for on-line linear scanning of human G-banded chromosomes quantitative analysis is described. This program was written for a Wang 720 C programmable desk calculator associated to the Zeiss scanning photometer MP01. The system can detect up to a maximum of 24 densitometric band peaks found along banded chromosome arms or chromatids, estimate the total arm length and localize bands in terms of their relative positions. The scanning stage under control is always returned to the pre-fixed scanning starting point (centromere) which allows a user to scan the specimen repeatedly at different sensitivities and thus to reject minor bands or suspected chromosome artifacts. This facilitates a better visualization of major bands and chromosome landmarks. A fully formated print-out on band localizations and their relative positions is obtained at the end of each scanning. The possibilities of the application of this program to band mapping of human chromosomes and to the study of small chromosome band aberrations is discussed. Human chromosome band mapping On-line analysis
Quantitative microscopy 2 K calculator
1. Introduction
Band localization
Interactive program
method-only offers a general idea concerning band size thus difficulting the recording and exchange of information and no data at all on its precise localization. In a later paper [3] we reviewed different procedures developed for chromosome scanning and we described a method for the precise localization o f Gand C-bands in human chromosomes based on measurements o f the relative position o f individual G- and C-bands carried out on high resolution densitometric tracings obtained by the scanning photomicrographs o f banded chromosomes. Photomicrographs offer the advantage that it is possible to change the object size and hence the scanning resolution. Errors introduced by the microscopist when localizing the centromere are thus reduced. This procedure improved our estimations o f relative band localizations. Chromosome no. 1 and the Y chromosome from normal individuals were used during our former pilot study. The application o f this m e t h o d also enabled us to draw a map for chromosome no. 1 representing relative band positions in both chromosome arms. Data thus obtained was compared with the nomenclature proposed in Paris [2] and chromosome land-
Previous research [ 1 ] allowed us to develop a new cytological procedure for inducing G-banding patterns in human chromosomes. We have also been able to draw a first qualitative map on the distribution o f major G-bands along the chromosome arms which was used for a more precise identification o f individual chromosome pairs. During the Paris Conference (1971) and in later meetings [2] the use o f a comparative map o f banding patterns obtained with different available banding procedures was proposed. A subdivision o f chromosome arms in regions according to specific bands or chromosome landmards was also suggested. This new chromosome nomenclature proved to be useful for description of chromosome aberrations and for experimental purposes. Despite the fact that chromosome arms were subdivided into regions and that a detailed description o f major bands and landmards was performed no quantitative data was reported applicable for accurate band localization along the ,chromosome arms. Moreover, during these meetings Q- and C-band size described as 'very small, small, intermediate, large and very large' was suggested to be expressed numerically from 1 - 5 . This 283
284
M.E. Drets, On-line scanning o f human banded chromosomes
•
•
-:
+:
+ I+L5
.:
TI.
Fig. 1. Diagram of a hypothetical banded chromosome analog densitometric tracing indicating the localization of 5 peaks• TL denotes total length of the chromosome arm. C and T denote centromere and telomere respectively• PL5 corresponds to the distance of densitometric peak 5 measured from the eentromere.
marks were re-defined in terms of their relative positions [3]. In our paper a 'band' was defined as any clearly detectable densitometric peak which appeared as a galvanometer deflection greater than 5% of the background in the same relative position in different individuals. 'Band' relative positions were obtained dividing the length of the position of the band or densitometric peak measured from the centromere on the tracing between the total length of the chromosome arm involved (fig. 1). This procedure of chromosome band mapping was confirmed by Mainguy et al. [4] who reported a similar method for precise band localization but based on measurements carried out on photographs of the chromosomes of the muntjak. Although measurements of analog densitometric tracings of banded chromosomes afford a precise and reliable way of chromosome band mapping it is a rather time consuming method since it is necessary to carry out many handmade measurements for estimating statistically relative positions on a high number of banded chromosomes before attempting to draw a reliable map. Photometers associated to high precision scanning microscope stages and small desk computers have lately become increasingly popular in laboratories of cytology. Obviously, the use of computers to the problem of band detection would save many hours of tiring measurements and would also permit to
obtain much more precise information which is fundamental for further statistical analysis. Computers have been extensively used to develop computational methods for chromosome identification, chromosome profiling and automated karyotyping [5-16]. A great number of these applications were carried out with large computers which are not always available to the cytogeneticist limiting their use to a reduced number of laboratories. It seems then, necessary to develop programs for minicomputers since they are most frequently associated to presently manufactured photometers and otherwise much easier to use. In the present paper, a computer program written for on-line interactive linear scanning of human banded chromosomes is described. This program estimates the total length of the chromosome arm and localizes bands in their relative positions. Since the user can select any sensitivity during each chromosome scanning, minor bands can be rejected helping him to visualize more easily chromosome landmarks or major bands. On the other hand, the possibility of including minor or variable bands may allow to make a much more precise statistical analysis of the normal distribution of bands and also to detect more accurately small chromosome band aberrations.
2. Instrumental set-up and cytological procedures BANDSCAN was developed for a Wang programmable calculator Model 720 C (app. 2 K) and output printer Model 701. This calculator has been associated by Carl Zeiss (Oberkochen) to their photometer system MP01. This instrumentation is composed of 4 electronic modules namely: measuring amplifier (part no. 477400-9901) and light modulator (50 cycles~s, part no. 474218); digital display unit (part no. 477443); stage control unit (part no. 477445) and interface for the Wang Calculator (part no. 477432). The photometer system was installed in a Zeiss Photomicroscope Model II and Luminar lenses (20 mm 1 : 3.5/0.15 N.A.; part no. 462513 and 40 mm 1:4.5/0.13 N.A.; part no. 462515) and planachromat 40 X 0.65 N.A., and planapochromat 100 × 1.3 N.A., oil, lenses (parts no. 460710 and no. 461940) were used for scanning photomicrographs and slides. Scanning stages (0.5/am steps; part no. 473481
285
M.E. Drets, On-line scanning of human banded chromosomes
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PRINT *
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. . . . . . .
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Fig. 2. Flowchart of the BANDSCAN program. Downward and upward curve slopes logics (arrows) are indicated for band no. 1 (see also inset). Dashed circles and line represent program logic from band no. 2 to band no. 24. After detection of 24 densitometric peaks the scanning stage completes the programmed scanning and returns to the starting point. 'Print results' logic appears in fig. 5.
286
M.E. Drets, On-line scanning of human banded chromosomes
and 10 jam steps; part no. 473482) were used indistinctly according to the size of the specimen scanned. Blood cultures of normal individuals, G-banding methods, slide processing and photographic procedures of banded chromosomes photomicrographs were reported elsewhere [3].
3. Computational procedures and program description 3.1. B A N D S C A N logic
BANDSCAN flowchart appears in fig. 2. Software was developed for an interactive densitometrical scanning analysis of G-banded human chromosomes slides or positives of photomicrographs of them. This description may serve as instructions for the use of the BANDSCAN program. Densitometric curves were considered as 'bands' [3] for the development of the software system, transmission lowest peaks representing characteristic points along chromosome arms (Fig. 1). Densitometric curves, expressed thus as transmission values measured by the digital unit were divided into two parts according to the downward or upward slope direction of the curve and they were used in the program logic (fig. 2 arrows and inset). Therefore, the computer first starts estimating decreasing values and storing lowest transmission values measured. When
................. ..... I "~' [: i~......... i"~ ............~i ................................................. /-"~ ~
Fig. 3. Diagram illustrating the logic followed for rejecting densitometric peaks. Scanning sensitivity (H) or desired curve height entered by the user is compared against height found during program execution. When a peak is rejected, the program continues estimating values in the corresponding curve slope. In case of a new curve inflection, the direction of the slope logic changes accordingly.
the lowest value of the series is found and a curve inflection occurs, the program continues in the following curve slope logic and the instrument starts thus calculating increasing values and storing the highest ones found. After a new curve inflection is detected, curve height - obtained by calct~lating the difference between A and B values - (see fig. 2) is compared against a curve height value (H) pre-fixed by the user. If A B is equal or greater than H, the lowest value and its localization length along the chromosome arm are stored. If not, the system erases data from the last slope logic continuing the calculations in the former one (fig. 3). This feature proved to be an effective filter for rejecting unwanted minor densitometric peaks or suspected artifacts facilitating the recognition of chromosome landmarks, major bands and the interpretation of the results obtained. Figure 4 shows a simplified flowchart of the peak filter or band rejection system for 2 successive bands. Inflection decision is based on the subtraction of 2 mean values calculated from 3 successive measurements. A change of sign indicates that a curve inflection has occurred. 3. 2. Program capacity
A maximum number of 24 densitometric peaks was considered to be sufficient for the study of banded human chromosome arms. In this regard, no more than 6 bands were described for the long arm of chromosome no. 1 (the largest one in the human complement) during the Paris Conference [2]. Moreover, previous studies on this chromosome arm carried out in our Laboratory (unpublished) showed that it is possible to detect consistently at least 10 bands in appropriate prometaphase banded chromosomes. Therefore, the possibility of analyzing up to 24 bands/human chromosome arm was considered a practical compromise between the rather reduced capacity of the computer (app. 2 K) and the chromosome banded structure. In case of an excessive high sensitivity and/or photographic grain irregularities, the machine will print * ADJ H (adjust H) and will complete the scanning returning to the scanning starting point without printing results (fig. 2 and fig. 8). Therefore, the cytogeneticist never loses the centromere position during chromosome scannings.
M.E. Drets, On-line scanning of human banded chromosomes
287
3. 3. Length chromosome and band measurement Scanning stage stepping was used for measuring, in artitrary units, the total chromosome arm length and for band localization. These values can be easily converted to actual distances in mm according to the stage used (0.5/lm step or 10 pm step). Pulses applied to the stepping motor were thus considered as length units in all calculations. BAND5
...g
BAND6
........
a
YtS
Fig. 4. Flowchart segment o f the BANDSCAN program showing the application o f the logic presented in fig. 3 used for
3.4. Instntment adjustment Since BANDSCAN was developed for linear scanning of chromosomes, they had to be oriented paralelly to the X-direction of stage movement of translation by means of a rotating specimen accessory (part no. 473490, modified). It should be pointed out that the computer will activate the stage to the observer's left after program starting. Therefore, this feature should be taken into account when positioning chromosomes for scanning and since the centromere is, in most of the cases, the scanning starting point it must be localized precisely. In the event that it wou.ld be preferred to start scanning from the telomere area the user must carry out trial scannings in order to locate the centromere and thus estimate the number of scanning steps to be used. An alternative method is to cut the photograph at the centromere region. In both cases, the program will effectively estimate the total arm length. However, due to the fact that the resulting relative band positions were measured from the telomere, the user must subtract values from 1 in order to obtain relative positions considered from the centromere. Slight variations 9f chromosome length detection (less than 0.1%) were related mainly to room temperature variations which produced minor changes in size both in the microscope stage and/or in the specimen itself. Notwithstanding, in adequate experimental conditions highly reproducible scannings were obtained (fig. 8). The system will compute and measure precisely chromosome structure only if a careful measurement
rejecting or including densitometric peaks. Logics for two successive bands (no. 5, 6) are included as an example. Arrows indicate upwards and downwards inclination of curve slope.
288
M.E. Drets, On-line scanning of human banded chromosomes
of the reference beam (BGND) is done. The best method was to measure the reference value in any area close to the chromosome to be scanned as, for instance, the area surrounding the telomere. Transmission reference value used as background can also be prefixed by the user adjusting both the amplification factor or the light intensity control unit (part no. 394030). A transmission value of 100 was found practical since overflow is allowed (Mode selector switch adjusted to T). Background measurements should, of course, be carried out after presetting photometer diaphragms, illumination, magnification,
high voltage and amplification. In order to 'integrate' whole bands and to avoid relative positions interpretation uncertainties produced by band distortions resulted from the denaturation banding procedure followed, rectangular diaphragms were used as luminous field stop (constructed at the Institute workshop) and as measuring stop (part no. 477350). Aperture of the luminous field stop at the plane of the specimen was at least three times the slit width of the measuring field diaphragm [20].
Table 1 BANDSCAN subroutines (SR) 720 C Wang code
Function
0000 0001 0002 0003 0004 0005 0006 0007 0008 0009 0010 0011 0012 0013 0014 0015
Splitting of registers SR - Store indirect halfA Splitting of registers SR - Recall indkect halfA Splitting of registers SR - Store indirect halfB Splitting of registers SR - Recall indkect half B Format Printout SR - Skips calculation and print results if X = 0 Recall band localization, calculate relative band position and recall total length Print results SR Stage control SR. Count number of stage steps and recall X length value (I) Stage control SR. Count number of stage steps and recall X length value (II) Repeat scan start SR Zeroes all operational and storage data registers Partial zeroing of operational data registers Partial zeroing of operational data registers SR for master routine (I) SR for master routine (II) BANDSCAN master routine. Program heading. Zeroing of all registers. Step length, X length, background and sensitivity storage. Starts program Background detection, transmission value storage (a) detection of lowest value Background detection, transmission value storage (b) detection of lowest value Store and localize lowest value Calculate mean value between (a) and (b) for downward curve slope Background detection, transmission value storage (c) detection of highest value Background detection, transmission value storage (d) detection of highest value Estimate curve height and compare it against H Calculate mean value between (c) and (d) for upward curve slope Step length counter SR Background SR Stage run and digital display measurement recall Stage scanning control (+X direction) Step length control SR Stage scanning control (-X direction) X step length control SR. Return to the starting point and print results at the end SR for master routine (I) SR for master routine (II)
0110 0111 0112 0113 0114 0115 0210 0211 0212 0214 0215 0310 0311 0312 0313 0314 0315
289
M.E. Drets, On-line scanning of human banded chromosomes 3.5. B A N D S C A N subroutines
BANDSCAN program was written in Wang machine language including 5 level subroutines. Table 1 describes subroutines used and their functions. Each subroutine is identified by the machine code. The program occupies the whole of the available core and due to the limited program storage size splitting of registers was used when possible ([ 17] modified). BANDSCAN analizer use was simplified to 2 program keyboard commands which start BANDSCAN master routine (0015) or repeat scan subroutine (0009). An additional command (0006) enables the user to obtain extra copies of the results printout. BANDSCAN master routine starts writing program heading. The user should then enter 4 values: step length (STEP); number of steps in the X-direction (X); reference signal values (measured or adjusted background) (BGND) and curve height (/-/). Repeat scan subroutine retains the 3 first values entered but the user should load again a new value for H. Repeat scan subroutine is identified by successive numbers each time it is used thus facilitating further data reference. The scanning stage is under full user's control but it should be noticed that a maximum length of 75 mm can be scanned with both models of scanning stages. Consequently, the combination of step length and number of steps should never exceed this value and photographic magnification should also fit to these dimensions. Number of steps are displayed continuously in the X-register, while in the Y-register appears the actual number of steps performed. All other photometer adjustments are carried out as usual with the MP01 photometer system. Additional information on these operations can be obtained in its operating manuals [ 18,19]. However, it must be noticed that the digital display unit averages results on the basis of 1 - 2 5 6 individual values (as a fraction of the AC cycling time) which reduces the background noise but increases the total time required to scan the specimen. In our scannings this switch was adjusted to 8 or 16. 3. 6 Printing results
Results are printed for each band detected along the chromosome arm as partial lengths (PL) (mea-
RECALk REGISTER
~ | , ~ |
PRINT TL
PRINT PL
CALCULATE RP
CRILF
SPACE 12
Fig. 5. 'Print results' format subroutine flowchart.
sured from the centromere in most of the cases) and relative positions (RP). The logic for the printing format and calculations of relative position is presented in fig. 5. Four columns of data (PL and RP) are printed in each line these values corresponding to each band detected. Finally, the total length of chromosome structure detected is printed, this value allowing the verification of the reproducibility of the scanning. Results obtained can be used either for plotting bands or for further statistical analysis. 3. 7. B A N D S C A N programming
BANDSCAN program can be keyed or transferred from magnetic tape to core and after pressing the Verify Program key the X-display should read 17909 (1551 steps) indicating that the program was correctly loaded. Program in execution can be halted when necessary simply pressing the Prime key which also resets the program counter to step 0000.
M.E. Drets, On-line scanning o f human banded chromosomes
290
4. Models used for BANDSCAN test BANDSCAN subroutines (Wang code no. 0010 and 0014) for storing the highest and lowest values of a series of numbers were first tested using increasing or decreasing values, respectively. Tests confirmed that these subroutines effectively stored these values. Crucial tests were carried out scanning a photograph of a drawing of a series of successive lines (fig. 6). These lines were drawn at different randomly selected locations. A random number generator was used for this line positioning and arbitrary values displayed were converted into millimeters. Drawing was photographed on High Contrast Film (Kodak) and developed in Microdol developer to minimize grains
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Fig. 6. Model used for testing BANDSCAN accuracy and dependability for detecting densitometric peaks. Left: Lines in the drawing were randomly positioned. Dotted lines indicate the scanning starting point. Expected values were calculated measuring distances in mm of each individual line from the starting point. Detected mean values were obtained after scanning 20 times a photographic reduction (X-12) of the original drawing through a Luminar lens (40 mm) and a 10 tam step stage. Slight differences between expected and detected values appearing for some lines were attributed to minor imperfections of line drawing and film grain, both magnified by the microscope system.
and increase resolution. A photographic positive reduction of this image was scanned with a Luminar lens (25 ram) and a scanning stage of 10/lm step. Figure 6 shows the close agreement found between expected and detected relative values. A final test was performed on a microscope object reticle (10/am). Planachromat lens (×40) and a 0.5 gm step stage were used. Imperfections in line engraving, occasionally present in these microscope reticles, were detected as differences in relative distances between lines (fig. 7). These imperfections, in our case, were estimated to range app. -+.1 gm.
5. Results and discussion Figures 8, 9 and 10 present some examples of BANDSCAN practical applications. Figure 8 is a sample run of the results print-out of a scanning of the long arm of a banded chromosome number 1 from a normal individual. An unadequate high sensitivity (H) was intentionally keyed-in in the first scanning. No results were printed in this case since the system had detected more than 24 peaks due to noise background and the user was advised to readjust H. Repeated scannings ( 1 - 4 ) shows how some bands were progressively rejected retaining only those major bands. Figure 9 shows a scanning of the same chromosome region from another person but data were ver-
M.E. Drets, On.line scanning of human banded chromosomes
291
BANDSCAN STEP 1 X 600 ~,lfl) 1 0 0 . 0 H .05 * ADJ 11
REPEAT SCAN H
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PL: PL: PL: PL: PL:
35 84 179 240 410
TL:
536
RP: liP: RP: RP: RP:
.0652 .1567 .3339 .4477 .7649
PL: PL: PL: PL: PL:
46 90 192 249 447
RP: RP: RP: RP: RP:
.0858 .1679 .3582 .4645 .8339
PL: PL: PL: PL: PL:
60 106 200 255 48~
RP: ~P: liP: RP: RP:
.1119 .1977 .3731 .4757 .9048
PL: PL: PL: PL: PL:
66 167 227 319 4q7
RP: RP: SP: RP: RP:
RP: .0858 RP: .3339 RP: . 6 7 9 I
PL: PL: PL:
60 192 410
RP: .1119 RP: .3582 RP: .7649
PL: PL: PL:
106 200 447
RP: .1977 RP: .3731 RP: .8339
PL: PL: PL:
167 319 497
RP: .3115 RP: .5951 RP: .9272
PL: PL:
106 497
RP: .1977 RP: .9272
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167
RP: .3115
PL:
319
RP: .5951
PL:
319
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PL:
410
RP: .7649
PL:
497
RP: .9272
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PL: PL: PL:
46 179 364
TL:
536
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Fig. 8. B A N D S C A N p r i n t o u t . L o n g a r m o f h u m a n c h r o m o s o m e n o . 1 w a s s c a n n e d at 5 d i f f e r e n t s c a n n i n g sensitivities (H). A n excessive h i g h s e n s i t i v i t y w a s u s e d first ( 0 . 0 5 ) d e t e c t i n g m o r e t h a n 2 4 d e n s i t o m e t r i c p e a k s . R e p e a t s c a n s u b r o u t i n e w a s t h e n u s e d at l o w e r sensitivities d u r i n g t h e f o l l o w i n g s c a n n i n g s ( 0 . 2 5 ; 0 . 5 0 ; 2 . 0 0 ; 5 . 0 0 ) . R e p e a t e d s c a n s are i n d i v i d u a l i z e d b y a c o u n t e r . N o t e t h a t in all cases t h e s y s t e m e s t i m a t e d t h e s a m e c h r o m o s o m e a r m l e n g t h . P a r t i a l l e n g t h s ( P L ) c o r r e s p o n d i n g to i n d i v i d u a l p e a k s , w e r e p r i n t e d t o g e t h e r w i t h t h e i r r e l a t i v e p o s i t i o n s ( R P ) in o r d e r t o f a c i l i t a t e t h e c y t o l o g i s t f u r t h e r d a t a r e f e r e n c e .
tically arranged according to the sensitivity used. At right, a diagram mapping of the bands detected is illustrated. In both cases, relative positions of chromosome landmarks and major bands accord with our
previously reported data [3]. FinaUy, fig. 10 illustrates 2 endoreduplicated G-banded human chromosomes no. 2, their relative band positions and the map obtained for each homo-
M.E. Drets, On-line scanning of human banded chromosomes
292
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Fig. 9. Diagrammatic representation of BANDSCAN results obtained at different scanning sensitivities and map obtained for the long arm of human chromosome no. 1. Minor bands are represented in the map by dashed lines.
logue. Interesting enough is the fact that several bands of these unusual chromosomes were located practically at the same relative position indicating the accuracy and reliability of the system. It has been already pointed out [21 ] the potential importance of maps of banded chromosomes representing bands in their relative positions. This method of chromosome identification is much more reliable than the use of a simple qualitative map of distribution of major bands based mainly on the observer's ability to detect them. Also, the application of such a quantitative map for each chromosome of the human complement would eventually allow the researcher and the clinician a better correlation between bands and gene mapping for instance, or other chromosome features since bands represent chromosome organization. Moreover, this map would be a more precise tool for studying minor chromosome changes related to human disease. In this regard, Marqallo et al. [22] have recently reported a
case of hemihypotrophy in a girl with a translocation t (13q; 7p). Photometric tracings of the translocated chromosome showed a 'surprising large wave' interpreted that 'may the new band be due to interaction between terminal 7p and proximal 13q chromatin'. In our opinion, the eventual application of quantitative computerized photometrical analysis on this chromosome taken as an example or on other band chromosome aberrations present in the human complement, would afford additional information for precise band localization. This would otherwise be of great interest for future reference on the human chromosome pathology and related congenital conditions, BANDSCAN applications can be easily expanded and band size or curve area for instance also estimated. Moreover, it is possible to use it for scanning negatives but subroutine order must be changed accordingly. This program proved to be useful for scanning R-banded chromosomes as well as banded chromosomes from other mammals.
M.E. Drets, On-line scanning of human banded chromosomes
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5rnu Fig. 10. Microphotograph of 2 endoreduplicated human chromosomes no. 2 homologues and diagrammatic representation of band relative positions as detected through the BANDSCAN program. A and B diagram chromosome halves represent each homologue. Note the agreement of some band values detected in both arms. 6. Mode
of
availability
The program listing is available from the author.
Acknowledgements The expert technical assistance o f B.R. de Olivera and G.A. Folle is acknowledged. The author is also grateful to G. Hornung (Carl Zeiss, Oberkochen) for useful suggestions on the Zeiss instrumentation. This work was supported in part by NIH GM 22037-02, WHO G3/181/8 5, OAS (RPDSAT) and Ministerio de Educaci6n y Cultura (Uruguay) grants.
References
[ 1 ] M.E. Drets and M.W. Shaw, Specific banding patterns of human chromosomes, Proc. Natl. Acad. Sci. USA 68 (1971) 2073-2077•
[2] Paris Conference (1971) Suppl. (1975) Standardization in human cytogenetics, ed. D. Bergsma, vol. XI, no. 9 pp. 1-36 (The National Foundation New York, 1975). [3] M.E. Drets and H. Seuanez, Quantitation of heterogeneous human heterochromatin: microdensitometric analysis of C- and G-bands, in: Physiology and Genetics of Reproduction, part A, eds. E.M. Coutinho and F. Fuchs, pp. 29-52 (Plenum Press, New York, 1975). [4] P.N.R. Mainguy, J.A. Heddle and D.M. Brunette, A reliable method of quantifying G-band position in chromosomes, Chromosoma (Berl.) 50 (1975) 301-312. [ 5 ] Automation of cytogenetics, Asilomar Workshop, Pacific Grove, Calif. ed. M.L. Mendelsohn pp. 1 - 187, Natl. Tech. Inf. Serv., US Department of Commerce, Springfield, VA, 1975). [6] J.W. Butler, M.K. Butler and A. Stroud, Automatic classification of chromosomes, Proc. Conf. Data Acquisition and Processing in Biology and Medicine, pp. 261275 (Pergamon Press, Oxford, 1963). [7] C. Disch~che and J. Bontemps, Method for the determination of mean densitometric profiles of chromosomes. Application to human chromosomes stained by Quinacrine mustard, ethidium bromide or by the
294
M.E. Drets, On4ine scanning of human banded chromosomes
Feulgen reaction, Chromosoma (Berl.) 54 (1976) 3 9 59. [81 C.J. Hilditch, The principles of a software system for karyotype analysis in: Human Population Cytogenetics, Univ. Edinburgh Pfizer Medical Monographs 5, eds. P.A. Jacobs, W.H. Price and P. Law, pp. 297-325 (Williams and Wilkins Co., Baltimore, 1970). [9] M.L. Mendelsohn, T.J. Conway, D.A. Hungerford, W.A. Kolman, B.H. Perry and J.M.S. Prewitt, Computeroriented analysis of human chromosomes. I. Photometric estimation of DNA content, Cytogenetics 5 (1966) 223-242. [10] M.L. Mendelsohn, D.A. Hungerford, B.H. Mayall, B. Perry, T. Conway and J.M.S. Prewitt, Computeroriented analysis of human chromosomes. II. Integrated optical density as a single parameter for karyotype analysis. Ann. NY Acad. Sci. 157 (1969) 376-392. [ 11 ] P.W. Neurath, B.L. Bablouzian, T.H. Warms, R.C. Serbagi and A. Falek, Human chromosome analysis by computer - an optical pattern recognition problem, Ann. NY Acad. Sci. 128 (1966) 1013-1028. [12] D. Rudovitz, J. Cameron, A.S.J. Farrow, R. Goldberg, D.K. Green and C.J. Hilditch, Instrumentation and organization Ibr chromosome measurement and karyotype analysis in: Human Population Cytogenetics, Univ. Edinburgh Pfizer Medical Monographs 5, eds. P.A. Jacobs, W.H. Price and P. Law, pp. 2 8 0 - 2 9 6 (Williams and Wilkins Co., Baltimore, 1970). [131 M. van der Ploeg, P. van Duijn and J.S. Ploem, Highresolution scanning-densitometry of photographic negatives of human metaphase chromosomes I. Instrumentation, Histochemistry 42 (1974) 9 - 2 9 .
[14] M. van der Ploeg, P. van Duijn and J.S. Ploem, Highresolution scanning-densitometry of photographic negatives of human metaphase chromosomes II. FeulgenDNA measurements, Histochemistry 42 (1974) 31-46. [15] G. von Stranzinger, H. Geldermann, P. Eberle and R. Maurer, Messpersonenvergleich an Humanchromosome mit Hilfe der halbautomatischen Chromosomenanalyse, Mikroskopie 27 (1971) 279-288. [16] N. Wald, R.W. Ranshaw, J.M. Herron and J.G. Castle, Progress on an automatic system for cytogenetic analysis, in: Human population cytogenetics, Univ. Univ. Edinburgh Pfizer Medical Monographs 5, eds. P.A. Jacobs, W.H. Price and P. Law, pp. 2 6 3 - 2 8 0 (Williams and Wilkins Co., Baltimore, 1970). [ 17 ] J. Gibson, Splitting of registers, 700 Series Programming Guide, pp. 5 0 - 5 3 (Wang Labs. Inc., 1970). [18] C. Zeiss, Microscope photometer 01 operating instructions, G 41-901,902,903 e, G 41-820 e (1971) 1-16. [19] C. Zeiss, MPM microscope photometer, G 40-830 e (1970) 1-27. [20 H.G. Zimmer, Microphotometry, Molecular Biology, Biochemistry and Biophysics Vol. 14 Micromethods in Molecular Biology, ed. V. Neuhoff, pp. 2 9 7 - 3 2 8 (Springer-Verlag, Berlin, 1973). [21] M.E. Drets, Human chromosome band mapping, The Lancet (1975) 1035. [22] F.A. Marqallo, L.C. Werneck, R.F. Pilotto and J.M. Opitz, Hemihypotrophy in a girl with a translocation t (13q; 7p) Eur. J. Pediat. 124 (1977) 167-171.
B A N D S C A N - A COMPUTER PROGRAM FOR ON-LINE LINEAR SCANNING OF HUMAN B A N D E D CHROMOSOMES Mfiximo E. DRETS Division of Human Cytogenetics, Instituto de lnvestigaciones Biol6gicas Clernente Estable, Avda. Italia 3318, Montevideo, Uruguay BANDSCAN, an interactive program for on-line linear scanning of human G-banded chromosomes quantitative analysis is described. This program was written for a Wang 720 C programmable desk calculator associated to the Zeiss scanning photometer MP01. The system can detect up to a maximum of 24 densitometric band peaks found along banded chromosome arms or chromatids, estimate the total arm length and localize bands in terms of their relative positions. The scanning stage under control is always returned to the pre-fixed scanning starting point (centromere) which allows a user to scan the specimen repeatedly at different sensitivities and thus to reject minor bands or suspected chromosome artifacts. This facilitates a better visualization of major bands and chromosome landmarks. A fully formated print-out on band localizations and their relative positions is obtained at the end of each scanning. The possibilities of the application of this program to band mapping of human chromosomes and to the study of small chromosome band aberrations is discussed. Human chromosome band mapping On-line analysis
Quantitative microscopy 2 K calculator
1. Introduction
Band localization
Interactive program
method-only offers a general idea concerning band size thus difficulting the recording and exchange of information and no data at all on its precise localization. In a later paper [3] we reviewed different procedures developed for chromosome scanning and we described a method for the precise localization o f Gand C-bands in human chromosomes based on measurements o f the relative position o f individual G- and C-bands carried out on high resolution densitometric tracings obtained by the scanning photomicrographs o f banded chromosomes. Photomicrographs offer the advantage that it is possible to change the object size and hence the scanning resolution. Errors introduced by the microscopist when localizing the centromere are thus reduced. This procedure improved our estimations o f relative band localizations. Chromosome no. 1 and the Y chromosome from normal individuals were used during our former pilot study. The application o f this m e t h o d also enabled us to draw a map for chromosome no. 1 representing relative band positions in both chromosome arms. Data thus obtained was compared with the nomenclature proposed in Paris [2] and chromosome land-
Previous research [ 1 ] allowed us to develop a new cytological procedure for inducing G-banding patterns in human chromosomes. We have also been able to draw a first qualitative map on the distribution o f major G-bands along the chromosome arms which was used for a more precise identification o f individual chromosome pairs. During the Paris Conference (1971) and in later meetings [2] the use o f a comparative map o f banding patterns obtained with different available banding procedures was proposed. A subdivision o f chromosome arms in regions according to specific bands or chromosome landmards was also suggested. This new chromosome nomenclature proved to be useful for description of chromosome aberrations and for experimental purposes. Despite the fact that chromosome arms were subdivided into regions and that a detailed description o f major bands and landmards was performed no quantitative data was reported applicable for accurate band localization along the ,chromosome arms. Moreover, during these meetings Q- and C-band size described as 'very small, small, intermediate, large and very large' was suggested to be expressed numerically from 1 - 5 . This 283
284
M.E. Drets, On-line scanning o f human banded chromosomes
•
•
-:
+:
+ I+L5
.:
TI.
Fig. 1. Diagram of a hypothetical banded chromosome analog densitometric tracing indicating the localization of 5 peaks• TL denotes total length of the chromosome arm. C and T denote centromere and telomere respectively• PL5 corresponds to the distance of densitometric peak 5 measured from the eentromere.
marks were re-defined in terms of their relative positions [3]. In our paper a 'band' was defined as any clearly detectable densitometric peak which appeared as a galvanometer deflection greater than 5% of the background in the same relative position in different individuals. 'Band' relative positions were obtained dividing the length of the position of the band or densitometric peak measured from the centromere on the tracing between the total length of the chromosome arm involved (fig. 1). This procedure of chromosome band mapping was confirmed by Mainguy et al. [4] who reported a similar method for precise band localization but based on measurements carried out on photographs of the chromosomes of the muntjak. Although measurements of analog densitometric tracings of banded chromosomes afford a precise and reliable way of chromosome band mapping it is a rather time consuming method since it is necessary to carry out many handmade measurements for estimating statistically relative positions on a high number of banded chromosomes before attempting to draw a reliable map. Photometers associated to high precision scanning microscope stages and small desk computers have lately become increasingly popular in laboratories of cytology. Obviously, the use of computers to the problem of band detection would save many hours of tiring measurements and would also permit to
obtain much more precise information which is fundamental for further statistical analysis. Computers have been extensively used to develop computational methods for chromosome identification, chromosome profiling and automated karyotyping [5-16]. A great number of these applications were carried out with large computers which are not always available to the cytogeneticist limiting their use to a reduced number of laboratories. It seems then, necessary to develop programs for minicomputers since they are most frequently associated to presently manufactured photometers and otherwise much easier to use. In the present paper, a computer program written for on-line interactive linear scanning of human banded chromosomes is described. This program estimates the total length of the chromosome arm and localizes bands in their relative positions. Since the user can select any sensitivity during each chromosome scanning, minor bands can be rejected helping him to visualize more easily chromosome landmarks or major bands. On the other hand, the possibility of including minor or variable bands may allow to make a much more precise statistical analysis of the normal distribution of bands and also to detect more accurately small chromosome band aberrations.
2. Instrumental set-up and cytological procedures BANDSCAN was developed for a Wang programmable calculator Model 720 C (app. 2 K) and output printer Model 701. This calculator has been associated by Carl Zeiss (Oberkochen) to their photometer system MP01. This instrumentation is composed of 4 electronic modules namely: measuring amplifier (part no. 477400-9901) and light modulator (50 cycles~s, part no. 474218); digital display unit (part no. 477443); stage control unit (part no. 477445) and interface for the Wang Calculator (part no. 477432). The photometer system was installed in a Zeiss Photomicroscope Model II and Luminar lenses (20 mm 1 : 3.5/0.15 N.A.; part no. 462513 and 40 mm 1:4.5/0.13 N.A.; part no. 462515) and planachromat 40 X 0.65 N.A., and planapochromat 100 × 1.3 N.A., oil, lenses (parts no. 460710 and no. 461940) were used for scanning photomicrographs and slides. Scanning stages (0.5/am steps; part no. 473481
285
M.E. Drets, On-line scanning of human banded chromosomes
I
[ERASEOA*A 1 I
I
M
,Es _
I i
'~
_
TYES
.....
BAN0 ~m'~I N" 24
PRINT *
ADd
H
YES
YES
/I I I
NEXT %~ IBAND i
. . . . . . .
IP-
Fig. 2. Flowchart of the BANDSCAN program. Downward and upward curve slopes logics (arrows) are indicated for band no. 1 (see also inset). Dashed circles and line represent program logic from band no. 2 to band no. 24. After detection of 24 densitometric peaks the scanning stage completes the programmed scanning and returns to the starting point. 'Print results' logic appears in fig. 5.
286
M.E. Drets, On-line scanning of human banded chromosomes
and 10 jam steps; part no. 473482) were used indistinctly according to the size of the specimen scanned. Blood cultures of normal individuals, G-banding methods, slide processing and photographic procedures of banded chromosomes photomicrographs were reported elsewhere [3].
3. Computational procedures and program description 3.1. B A N D S C A N logic
BANDSCAN flowchart appears in fig. 2. Software was developed for an interactive densitometrical scanning analysis of G-banded human chromosomes slides or positives of photomicrographs of them. This description may serve as instructions for the use of the BANDSCAN program. Densitometric curves were considered as 'bands' [3] for the development of the software system, transmission lowest peaks representing characteristic points along chromosome arms (Fig. 1). Densitometric curves, expressed thus as transmission values measured by the digital unit were divided into two parts according to the downward or upward slope direction of the curve and they were used in the program logic (fig. 2 arrows and inset). Therefore, the computer first starts estimating decreasing values and storing lowest transmission values measured. When
................. ..... I "~' [: i~......... i"~ ............~i ................................................. /-"~ ~
Fig. 3. Diagram illustrating the logic followed for rejecting densitometric peaks. Scanning sensitivity (H) or desired curve height entered by the user is compared against height found during program execution. When a peak is rejected, the program continues estimating values in the corresponding curve slope. In case of a new curve inflection, the direction of the slope logic changes accordingly.
the lowest value of the series is found and a curve inflection occurs, the program continues in the following curve slope logic and the instrument starts thus calculating increasing values and storing the highest ones found. After a new curve inflection is detected, curve height - obtained by calct~lating the difference between A and B values - (see fig. 2) is compared against a curve height value (H) pre-fixed by the user. If A B is equal or greater than H, the lowest value and its localization length along the chromosome arm are stored. If not, the system erases data from the last slope logic continuing the calculations in the former one (fig. 3). This feature proved to be an effective filter for rejecting unwanted minor densitometric peaks or suspected artifacts facilitating the recognition of chromosome landmarks, major bands and the interpretation of the results obtained. Figure 4 shows a simplified flowchart of the peak filter or band rejection system for 2 successive bands. Inflection decision is based on the subtraction of 2 mean values calculated from 3 successive measurements. A change of sign indicates that a curve inflection has occurred. 3. 2. Program capacity
A maximum number of 24 densitometric peaks was considered to be sufficient for the study of banded human chromosome arms. In this regard, no more than 6 bands were described for the long arm of chromosome no. 1 (the largest one in the human complement) during the Paris Conference [2]. Moreover, previous studies on this chromosome arm carried out in our Laboratory (unpublished) showed that it is possible to detect consistently at least 10 bands in appropriate prometaphase banded chromosomes. Therefore, the possibility of analyzing up to 24 bands/human chromosome arm was considered a practical compromise between the rather reduced capacity of the computer (app. 2 K) and the chromosome banded structure. In case of an excessive high sensitivity and/or photographic grain irregularities, the machine will print * ADJ H (adjust H) and will complete the scanning returning to the scanning starting point without printing results (fig. 2 and fig. 8). Therefore, the cytogeneticist never loses the centromere position during chromosome scannings.
M.E. Drets, On-line scanning of human banded chromosomes
287
3. 3. Length chromosome and band measurement Scanning stage stepping was used for measuring, in artitrary units, the total chromosome arm length and for band localization. These values can be easily converted to actual distances in mm according to the stage used (0.5/lm step or 10 pm step). Pulses applied to the stepping motor were thus considered as length units in all calculations. BAND5
...g
BAND6
........
a
YtS
Fig. 4. Flowchart segment o f the BANDSCAN program showing the application o f the logic presented in fig. 3 used for
3.4. Instntment adjustment Since BANDSCAN was developed for linear scanning of chromosomes, they had to be oriented paralelly to the X-direction of stage movement of translation by means of a rotating specimen accessory (part no. 473490, modified). It should be pointed out that the computer will activate the stage to the observer's left after program starting. Therefore, this feature should be taken into account when positioning chromosomes for scanning and since the centromere is, in most of the cases, the scanning starting point it must be localized precisely. In the event that it wou.ld be preferred to start scanning from the telomere area the user must carry out trial scannings in order to locate the centromere and thus estimate the number of scanning steps to be used. An alternative method is to cut the photograph at the centromere region. In both cases, the program will effectively estimate the total arm length. However, due to the fact that the resulting relative band positions were measured from the telomere, the user must subtract values from 1 in order to obtain relative positions considered from the centromere. Slight variations 9f chromosome length detection (less than 0.1%) were related mainly to room temperature variations which produced minor changes in size both in the microscope stage and/or in the specimen itself. Notwithstanding, in adequate experimental conditions highly reproducible scannings were obtained (fig. 8). The system will compute and measure precisely chromosome structure only if a careful measurement
rejecting or including densitometric peaks. Logics for two successive bands (no. 5, 6) are included as an example. Arrows indicate upwards and downwards inclination of curve slope.
288
M.E. Drets, On-line scanning of human banded chromosomes
of the reference beam (BGND) is done. The best method was to measure the reference value in any area close to the chromosome to be scanned as, for instance, the area surrounding the telomere. Transmission reference value used as background can also be prefixed by the user adjusting both the amplification factor or the light intensity control unit (part no. 394030). A transmission value of 100 was found practical since overflow is allowed (Mode selector switch adjusted to T). Background measurements should, of course, be carried out after presetting photometer diaphragms, illumination, magnification,
high voltage and amplification. In order to 'integrate' whole bands and to avoid relative positions interpretation uncertainties produced by band distortions resulted from the denaturation banding procedure followed, rectangular diaphragms were used as luminous field stop (constructed at the Institute workshop) and as measuring stop (part no. 477350). Aperture of the luminous field stop at the plane of the specimen was at least three times the slit width of the measuring field diaphragm [20].
Table 1 BANDSCAN subroutines (SR) 720 C Wang code
Function
0000 0001 0002 0003 0004 0005 0006 0007 0008 0009 0010 0011 0012 0013 0014 0015
Splitting of registers SR - Store indirect halfA Splitting of registers SR - Recall indkect halfA Splitting of registers SR - Store indirect halfB Splitting of registers SR - Recall indkect half B Format Printout SR - Skips calculation and print results if X = 0 Recall band localization, calculate relative band position and recall total length Print results SR Stage control SR. Count number of stage steps and recall X length value (I) Stage control SR. Count number of stage steps and recall X length value (II) Repeat scan start SR Zeroes all operational and storage data registers Partial zeroing of operational data registers Partial zeroing of operational data registers SR for master routine (I) SR for master routine (II) BANDSCAN master routine. Program heading. Zeroing of all registers. Step length, X length, background and sensitivity storage. Starts program Background detection, transmission value storage (a) detection of lowest value Background detection, transmission value storage (b) detection of lowest value Store and localize lowest value Calculate mean value between (a) and (b) for downward curve slope Background detection, transmission value storage (c) detection of highest value Background detection, transmission value storage (d) detection of highest value Estimate curve height and compare it against H Calculate mean value between (c) and (d) for upward curve slope Step length counter SR Background SR Stage run and digital display measurement recall Stage scanning control (+X direction) Step length control SR Stage scanning control (-X direction) X step length control SR. Return to the starting point and print results at the end SR for master routine (I) SR for master routine (II)
0110 0111 0112 0113 0114 0115 0210 0211 0212 0214 0215 0310 0311 0312 0313 0314 0315
289
M.E. Drets, On-line scanning of human banded chromosomes 3.5. B A N D S C A N subroutines
BANDSCAN program was written in Wang machine language including 5 level subroutines. Table 1 describes subroutines used and their functions. Each subroutine is identified by the machine code. The program occupies the whole of the available core and due to the limited program storage size splitting of registers was used when possible ([ 17] modified). BANDSCAN analizer use was simplified to 2 program keyboard commands which start BANDSCAN master routine (0015) or repeat scan subroutine (0009). An additional command (0006) enables the user to obtain extra copies of the results printout. BANDSCAN master routine starts writing program heading. The user should then enter 4 values: step length (STEP); number of steps in the X-direction (X); reference signal values (measured or adjusted background) (BGND) and curve height (/-/). Repeat scan subroutine retains the 3 first values entered but the user should load again a new value for H. Repeat scan subroutine is identified by successive numbers each time it is used thus facilitating further data reference. The scanning stage is under full user's control but it should be noticed that a maximum length of 75 mm can be scanned with both models of scanning stages. Consequently, the combination of step length and number of steps should never exceed this value and photographic magnification should also fit to these dimensions. Number of steps are displayed continuously in the X-register, while in the Y-register appears the actual number of steps performed. All other photometer adjustments are carried out as usual with the MP01 photometer system. Additional information on these operations can be obtained in its operating manuals [ 18,19]. However, it must be noticed that the digital display unit averages results on the basis of 1 - 2 5 6 individual values (as a fraction of the AC cycling time) which reduces the background noise but increases the total time required to scan the specimen. In our scannings this switch was adjusted to 8 or 16. 3. 6 Printing results
Results are printed for each band detected along the chromosome arm as partial lengths (PL) (mea-
RECALk REGISTER
~ | , ~ |
PRINT TL
PRINT PL
CALCULATE RP
CRILF
SPACE 12
Fig. 5. 'Print results' format subroutine flowchart.
sured from the centromere in most of the cases) and relative positions (RP). The logic for the printing format and calculations of relative position is presented in fig. 5. Four columns of data (PL and RP) are printed in each line these values corresponding to each band detected. Finally, the total length of chromosome structure detected is printed, this value allowing the verification of the reproducibility of the scanning. Results obtained can be used either for plotting bands or for further statistical analysis. 3. 7. B A N D S C A N programming
BANDSCAN program can be keyed or transferred from magnetic tape to core and after pressing the Verify Program key the X-display should read 17909 (1551 steps) indicating that the program was correctly loaded. Program in execution can be halted when necessary simply pressing the Prime key which also resets the program counter to step 0000.
M.E. Drets, On-line scanning o f human banded chromosomes
290
4. Models used for BANDSCAN test BANDSCAN subroutines (Wang code no. 0010 and 0014) for storing the highest and lowest values of a series of numbers were first tested using increasing or decreasing values, respectively. Tests confirmed that these subroutines effectively stored these values. Crucial tests were carried out scanning a photograph of a drawing of a series of successive lines (fig. 6). These lines were drawn at different randomly selected locations. A random number generator was used for this line positioning and arbitrary values displayed were converted into millimeters. Drawing was photographed on High Contrast Film (Kodak) and developed in Microdol developer to minimize grains
EXPECTED .0800
~
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. 1200
///I1.1500 .ZIUU
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,~
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2.0007 ±.0006
2308
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2.0004
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.,ooo
5747
Fig. 7. Microscope stage reticle (10 tam) scanned with BANDSCAN to detect imperfections of line engraving separation distances. The reticle was scanned with a 0.5 tam step stage and a Planachromat (×40 lens). Values are indicated in relative units and they were placed approximately where the reticle was scanned. Relative distance of 524 represented probably a separation of l0 tam between lines which the manufacturer intended to engrave.
STD DEV.
.08oo
2300
. . . . . . . . . ~8oo
~F""
DETECTED (Mean)
Ft4t5oto!5417f 3121
2.0003
Fig. 6. Model used for testing BANDSCAN accuracy and dependability for detecting densitometric peaks. Left: Lines in the drawing were randomly positioned. Dotted lines indicate the scanning starting point. Expected values were calculated measuring distances in mm of each individual line from the starting point. Detected mean values were obtained after scanning 20 times a photographic reduction (X-12) of the original drawing through a Luminar lens (40 mm) and a 10 tam step stage. Slight differences between expected and detected values appearing for some lines were attributed to minor imperfections of line drawing and film grain, both magnified by the microscope system.
and increase resolution. A photographic positive reduction of this image was scanned with a Luminar lens (25 ram) and a scanning stage of 10/lm step. Figure 6 shows the close agreement found between expected and detected relative values. A final test was performed on a microscope object reticle (10/am). Planachromat lens (×40) and a 0.5 gm step stage were used. Imperfections in line engraving, occasionally present in these microscope reticles, were detected as differences in relative distances between lines (fig. 7). These imperfections, in our case, were estimated to range app. -+.1 gm.
5. Results and discussion Figures 8, 9 and 10 present some examples of BANDSCAN practical applications. Figure 8 is a sample run of the results print-out of a scanning of the long arm of a banded chromosome number 1 from a normal individual. An unadequate high sensitivity (H) was intentionally keyed-in in the first scanning. No results were printed in this case since the system had detected more than 24 peaks due to noise background and the user was advised to readjust H. Repeated scannings ( 1 - 4 ) shows how some bands were progressively rejected retaining only those major bands. Figure 9 shows a scanning of the same chromosome region from another person but data were ver-
M.E. Drets, On.line scanning of human banded chromosomes
291
BANDSCAN STEP 1 X 600 ~,lfl) 1 0 0 . 0 H .05 * ADJ 11
REPEAT SCAN H
.25
PL: PL: PL: PL: PL:
35 84 179 240 410
TL:
536
RP: liP: RP: RP: RP:
.0652 .1567 .3339 .4477 .7649
PL: PL: PL: PL: PL:
46 90 192 249 447
RP: RP: RP: RP: RP:
.0858 .1679 .3582 .4645 .8339
PL: PL: PL: PL: PL:
60 106 200 255 48~
RP: ~P: liP: RP: RP:
.1119 .1977 .3731 .4757 .9048
PL: PL: PL: PL: PL:
66 167 227 319 4q7
RP: RP: SP: RP: RP:
RP: .0858 RP: .3339 RP: . 6 7 9 I
PL: PL: PL:
60 192 410
RP: .1119 RP: .3582 RP: .7649
PL: PL: PL:
106 200 447
RP: .1977 RP: .3731 RP: .8339
PL: PL: PL:
167 319 497
RP: .3115 RP: .5951 RP: .9272
PL: PL:
106 497
RP: .1977 RP: .9272
PL:
167
RP: .3115
PL:
319
RP: .5951
PL:
319
RP: .5951
PL:
410
RP: .7649
PL:
497
RP: .9272
REPEAT SCAN II
.50
PL: PL: PL:
46 179 364
TL:
536
REPEAT SCAN ]1
.1231 .3115 .4235 .5951 .9272
2
3
2.00
PL: PL:
60 41C
TL:
536
RP: . l U 9 RP: .7649
REPEAT SCAN H
1
4
5.00
PL:
60
TL:
536
RP: .1119
Fig. 8. B A N D S C A N p r i n t o u t . L o n g a r m o f h u m a n c h r o m o s o m e n o . 1 w a s s c a n n e d at 5 d i f f e r e n t s c a n n i n g sensitivities (H). A n excessive h i g h s e n s i t i v i t y w a s u s e d first ( 0 . 0 5 ) d e t e c t i n g m o r e t h a n 2 4 d e n s i t o m e t r i c p e a k s . R e p e a t s c a n s u b r o u t i n e w a s t h e n u s e d at l o w e r sensitivities d u r i n g t h e f o l l o w i n g s c a n n i n g s ( 0 . 2 5 ; 0 . 5 0 ; 2 . 0 0 ; 5 . 0 0 ) . R e p e a t e d s c a n s are i n d i v i d u a l i z e d b y a c o u n t e r . N o t e t h a t in all cases t h e s y s t e m e s t i m a t e d t h e s a m e c h r o m o s o m e a r m l e n g t h . P a r t i a l l e n g t h s ( P L ) c o r r e s p o n d i n g to i n d i v i d u a l p e a k s , w e r e p r i n t e d t o g e t h e r w i t h t h e i r r e l a t i v e p o s i t i o n s ( R P ) in o r d e r t o f a c i l i t a t e t h e c y t o l o g i s t f u r t h e r d a t a r e f e r e n c e .
tically arranged according to the sensitivity used. At right, a diagram mapping of the bands detected is illustrated. In both cases, relative positions of chromosome landmarks and major bands accord with our
previously reported data [3]. FinaUy, fig. 10 illustrates 2 endoreduplicated G-banded human chromosomes no. 2, their relative band positions and the map obtained for each homo-
M.E. Drets, On-line scanning of human banded chromosomes
292
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logue. Interesting enough is the fact that several bands of these unusual chromosomes were located practically at the same relative position indicating the accuracy and reliability of the system. It has been already pointed out [21 ] the potential importance of maps of banded chromosomes representing bands in their relative positions. This method of chromosome identification is much more reliable than the use of a simple qualitative map of distribution of major bands based mainly on the observer's ability to detect them. Also, the application of such a quantitative map for each chromosome of the human complement would eventually allow the researcher and the clinician a better correlation between bands and gene mapping for instance, or other chromosome features since bands represent chromosome organization. Moreover, this map would be a more precise tool for studying minor chromosome changes related to human disease. In this regard, Marqallo et al. [22] have recently reported a
case of hemihypotrophy in a girl with a translocation t (13q; 7p). Photometric tracings of the translocated chromosome showed a 'surprising large wave' interpreted that 'may the new band be due to interaction between terminal 7p and proximal 13q chromatin'. In our opinion, the eventual application of quantitative computerized photometrical analysis on this chromosome taken as an example or on other band chromosome aberrations present in the human complement, would afford additional information for precise band localization. This would otherwise be of great interest for future reference on the human chromosome pathology and related congenital conditions, BANDSCAN applications can be easily expanded and band size or curve area for instance also estimated. Moreover, it is possible to use it for scanning negatives but subroutine order must be changed accordingly. This program proved to be useful for scanning R-banded chromosomes as well as banded chromosomes from other mammals.
M.E. Drets, On-line scanning of human banded chromosomes
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of
availability
The program listing is available from the author.
Acknowledgements The expert technical assistance o f B.R. de Olivera and G.A. Folle is acknowledged. The author is also grateful to G. Hornung (Carl Zeiss, Oberkochen) for useful suggestions on the Zeiss instrumentation. This work was supported in part by NIH GM 22037-02, WHO G3/181/8 5, OAS (RPDSAT) and Ministerio de Educaci6n y Cultura (Uruguay) grants.
References
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[2] Paris Conference (1971) Suppl. (1975) Standardization in human cytogenetics, ed. D. Bergsma, vol. XI, no. 9 pp. 1-36 (The National Foundation New York, 1975). [3] M.E. Drets and H. Seuanez, Quantitation of heterogeneous human heterochromatin: microdensitometric analysis of C- and G-bands, in: Physiology and Genetics of Reproduction, part A, eds. E.M. Coutinho and F. Fuchs, pp. 29-52 (Plenum Press, New York, 1975). [4] P.N.R. Mainguy, J.A. Heddle and D.M. Brunette, A reliable method of quantifying G-band position in chromosomes, Chromosoma (Berl.) 50 (1975) 301-312. [ 5 ] Automation of cytogenetics, Asilomar Workshop, Pacific Grove, Calif. ed. M.L. Mendelsohn pp. 1 - 187, Natl. Tech. Inf. Serv., US Department of Commerce, Springfield, VA, 1975). [6] J.W. Butler, M.K. Butler and A. Stroud, Automatic classification of chromosomes, Proc. Conf. Data Acquisition and Processing in Biology and Medicine, pp. 261275 (Pergamon Press, Oxford, 1963). [7] C. Disch~che and J. Bontemps, Method for the determination of mean densitometric profiles of chromosomes. Application to human chromosomes stained by Quinacrine mustard, ethidium bromide or by the
294
M.E. Drets, On4ine scanning of human banded chromosomes
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[14] M. van der Ploeg, P. van Duijn and J.S. Ploem, Highresolution scanning-densitometry of photographic negatives of human metaphase chromosomes II. FeulgenDNA measurements, Histochemistry 42 (1974) 31-46. [15] G. von Stranzinger, H. Geldermann, P. Eberle and R. Maurer, Messpersonenvergleich an Humanchromosome mit Hilfe der halbautomatischen Chromosomenanalyse, Mikroskopie 27 (1971) 279-288. [16] N. Wald, R.W. Ranshaw, J.M. Herron and J.G. Castle, Progress on an automatic system for cytogenetic analysis, in: Human population cytogenetics, Univ. Univ. Edinburgh Pfizer Medical Monographs 5, eds. P.A. Jacobs, W.H. Price and P. Law, pp. 2 6 3 - 2 8 0 (Williams and Wilkins Co., Baltimore, 1970). [ 17 ] J. Gibson, Splitting of registers, 700 Series Programming Guide, pp. 5 0 - 5 3 (Wang Labs. Inc., 1970). [18] C. Zeiss, Microscope photometer 01 operating instructions, G 41-901,902,903 e, G 41-820 e (1971) 1-16. [19] C. Zeiss, MPM microscope photometer, G 40-830 e (1970) 1-27. [20 H.G. Zimmer, Microphotometry, Molecular Biology, Biochemistry and Biophysics Vol. 14 Micromethods in Molecular Biology, ed. V. Neuhoff, pp. 2 9 7 - 3 2 8 (Springer-Verlag, Berlin, 1973). [21] M.E. Drets, Human chromosome band mapping, The Lancet (1975) 1035. [22] F.A. Marqallo, L.C. Werneck, R.F. Pilotto and J.M. Opitz, Hemihypotrophy in a girl with a translocation t (13q; 7p) Eur. J. Pediat. 124 (1977) 167-171.