UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The effect of misonidazole on cell survival at low doses of radiation Faddegon, Bruce Alan 1983

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-UBC_1983_A6_7 F32.pdf [ 6.61MB ]
Metadata
JSON: 831-1.0085780.json
JSON-LD: 831-1.0085780-ld.json
RDF/XML (Pretty): 831-1.0085780-rdf.xml
RDF/JSON: 831-1.0085780-rdf.json
Turtle: 831-1.0085780-turtle.txt
N-Triples: 831-1.0085780-rdf-ntriples.txt
Original Record: 831-1.0085780-source.json
Full Text
831-1.0085780-fulltext.txt
Citation
831-1.0085780.ris

Full Text

THE EFFECT OF MISONIDAZOLE ON CELL SURVIVAL AT LOW DOSES OF RADIATION ;/; by BRUCE ALAN FADDEGON B.Sc., University of V i c t o r i a , 1977 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES THE DEPARTMENT OF PHYSICS We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May 1983 (c) Bruce Alan Faddegon, 1983 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of PHYSICS The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date May 10, 1983. )E-6 (.3/81) ABSTRACT Since Puck and Marcus f i r s t measured the effect of ionizing radiation on mammalian c e l l survival in 1956 the 'Puck plating' assay has been widely used as an endpoint for radiobiology experiments. The assay has contributed greatly to our knowledge of the processes involved when radiation interacts with c e l l s , tissues, and whole animals. Gener.ally c e l l survival experiments are carried out at f a i r l y high doses of radiation (5-30 Gray) where c e l l survival drops below 50% of the survival of un-irradiated c e l l s . Very l i t t l e data has been accumulated at lower doses. Most of our knowledge in this region has been extrapolated from measurements made at higher doses. This i s in part due to the d i f f i c u l t y of obtaining accurate results at higher survival levels, a problem primarily due to the d i f f i c u l t y in accurately determining the number of cells plated for the assay. This s t a t i s t i c a l uncertainty becomes important at survival levels greater than 50%. The uncertainty can be significantly reduced at these survival levels by accurately counting the number of cells plated, using a microscope, a procedure which i s very time consuming when performed manually. A method of automating the procedure with a computer controlled motor driven microscope stage i s described in this thesis. With the automated procedure the assay time i s reduced by a factor of three. In this thesis the effect of the radiosensitizer misonidazole on c e l l survival was measured at radiation doses as low as one Gray. The results , show that the drug i s a poor radiosensitizer at low doses for the experimental system used. The c l i n i c a l and radiobiological significance of these observations i s discussed. This experiment exemplifies the value of measuring c e l l survival at low doses. i i TABLE OF CONTENTS PAGE Abstract i i Table of contents i i i L i s t of tables v i L i s t of figures v i i L i s t of equations v i i i L i s t of abbreviations and glossary of terms i x Acknowledgements . . . . x i INTRODUCTION AND HISTORICAL REVIEW. 1 1.1 The science of radiobiology 1 1.2 The i n t e r a c t i o n of i o n i z i n g r a d i a t i o n with c e l l s 1 1.2.1 Quantifying e f f e c t s with the conventional assay of c e l l s u r v i v a l 3 1.2.2 Measurements of c e l l s u r v i v a l at low doses of i o n i z i n g r a d i a t i o n 7 1.2.3 Case i n point f o r low dose studies: the oxygen e f f e c t . . . 9 MATERIALS AND METHODS. 13 2.1 C e l l culture procedures 13 2.2 I r r a d i a t i o n procedures 13 2.3 C e l l s u r v i v a l assays 16 2.3.1 The conventional assay 16 2.3.2 The low dose assay 18 i i i TABLE OF CONTENTS (continued) PAGE 2.4 The automated low dose assay system, ALDAS 20 2.4.1 ALDAS hardware 22 2.4.2 ALDAS software • 27 2.4.3 The c e l l l o c a t i n g program LDALOC 31 2.4.4 The c e l l c l a s s i f i c a t i o n program LDACLASS 36 PURPOSE AND PROCEDURE. 41 3.1 Purpose of the experiments 41 3.2 Experiment set A 45 3.2.1 Purpose and procedure 45 3.2.2 Choosing between the low dose and conventional assays . . . 47 3.4.3 S u r v i v a l data 47 3.3 Experiment set B 48 3.3.1 Motivation f o r an improved low dose assay procedure . . . . 48 3.3.2 ALDAS and the low dose assay 50 3.3.3 Purpose and procedure 53 3.3.4 Survival data 55 RESULTS AND DISCUSSION. 57 4.1 Uncertainties 57 4.1.1 Experimental error, b i o l o g i c a l v a r i a t i o n , and experimenter er r o r 57 4.1.2 Radiation dose rate and drug concentration u n c e r t a i n t i e s . . 58 4.1.3 Survival assay unc e r t a i n t i e s 59 4.1.4 The t o t a l uncertainty i n s u r v i v a l 63 iv TABLE OF CONTENTS (continued) PAGE 4.2 Results 66 4.2.1 Method of parameter estimation 66 4.2.2 Method of confidence estimation 69 4.2.3 Results of parameter and confidence estimates 72 4.3 Signi f i c a n c e of the r e s u l t s 79 4.3.1 Misonidazole DMF dependence on s u r v i v a l l e v e l 83 4.3.2 C l i n i c a l s i g n i f i c a n c e of the r e s u l t s 86 4.3.3 Radiob i o l o g i c a l s i g n i f i c a n c e of the r e s u l t s : the molecular theory of c e l l s u r v i v a l 87 4.3.4 Suggested improvements to the experimental method 88 4.4 Su r v i v a l measurements at low doses 90 4.5 Future research 92 4.5.1 Discrepency i n s u r v i v a l measurements between assays . . . . 92 4.5.2 Survival dependence on oxygen tension 93 4.5.3 Observation of c e l l u l a r growth and d i v i s i o n 93 4.5.4 Su r v i v a l measurements at low doses of i o n i z i n g r a d i a t i o n . . 93 4.6 Summary 94 BIBLIOGRAPHY. 95 APPENDIX. L i s t i n g s of the ALDAS software routines 101 v LIST OF TABLES PAGE I L i s t of the components of ALDAS 28 II Normalized, averaged, s u r v i v a l data, experiment set B 56 III Chi-squared f i t r e s u l t s 70 IV Summary of experiment sets A and B r e s u l t s f o r alpha and beta. . . 82 V DMF at s u r v i v a l l e v e l s of 0.01, 0.1, 0.5, and 0.8, experiment set B 85 VI S p e c i f i c a t i o n s of the automated low dose assay system, ALDAS . . . 91 v i LIST OF FIGURES PAGE 1 Surv i v a l curve showing the oxygen e f f e c t 5 2 Oxygen enhancement r a t i o at low doses of r a d i a t i o n 12 3 The f l a s k s used f o r growing and i r r a d i a t i n g c e l l s 14 4 Diagram of the i r r a d i a t i o n setup 15 5 Comparison of the c e l l s u r v i v a l assay procedures 17 6 LDA l o c a t i o n chart 21 7 Photographs of ALDAS 23 8 Schematic of ALDAS 24 9 Schematic of the microscope stage d r i v e r 26 10 I/O ports on analog to d i g i t a l converter 29 11 LDALOC flowchart 32 12 LDACLASS flowchart 37 13 Time course of experiments set B 54 14 Alpha and beta confidence contours experiment set A 75 15 Alpha and beta 70% confidence contours experiment set B 76 16 Alpha and beta 95% confidence contours experiment set B 77 17 Alpha and beta 99.9% confidence contours, experiment set B . . . . 78 18 Alpha and beta dependence on misonidazole, experiment set A. . . . 80 19 Alpha and beta dependence on misonidazole, experiment set B. . . . 81 20 Sur v i v a l curves, experiment set B 84 v i i LIST OF EQUATIONS PAGE 1 Conventional assay s u r v i v a l c a l c u l a t i o n 16 2 Low dose assay s u r v i v a l c a l c u l a t i o n 20 3 Linear-quadratic s u r v i v a l equation 44 4 Standard deviation of the binomial d i s t r i b u t i o n 59 5 Low dose assay standard deviation i n s u r v i v a l 60 6 Standard deviation of the Poisson d i s t r i b u t i o n 60 7 Conventional assay standard deviation of the colony count 61 8 Conventional assay standard deviation i n s u r v i v a l 61 9 Conventional assay, s i m p l i f i c a t i o n of the standard deviation i n s u r v i v a l 61 10 Low dose assay minimum standard deviation i n s u r v i v a l with the uncertainty i n p l a t i n g e f f i c i e n c y taken i n t o account 62 11 Conventional assay minimum standard deviation i n s u r v i v a l with the uncertainty i n p l a t i n g e f f i c i e n c y taken i n t o account 62 12 The r a t i o i n the number of c e l l s to be plated f o r the low dose and conventional assays to achieve the same accuracy i n s u r v i v a l measurement 63 13 Estimate i n the standard deviation of s u r v i v a l from s u r v i v a l measurements 64 14 Estimate i n the standard deviation of s u r v i v a l from uncertainty estimates 64 15 The chi-squared equation 65 16 The re s i d u a l s (mathematical d e f i n i t i o n ) 65 17 Estimate of standard deviation of s u r v i v a l from a sing l e measure-ment of s u r v i v a l 66 18 A s i m p l i f i e d chi-squared equation 68 v i i i LIST OF ABBREVIATIONS AND GLOSSARY OF TERMS A/D Analog to d i g i t a l conversion. ALPAS The automated low dose assay system, section 2.4. ASCAN ALDAS subroutine used f o r moving the microscope stage, section 2.4.2. CHO c e l l s Chinese hamster ovary c e l l s . c l a s s i f i c a t i o n day The day on which c e l l s are c l a s s i f i e d when using the low dose assay. This i s generally 6-8 days following i r r a d i a t i o n . coincidence The c l a s s i f i c a t i o n used f o r the low dose assay when two colonies overlap on c l a s s i f i c a t i o n day. conventional assay The c e l l s u r v i v a l assay used i n t h i s study when i t i s estimated that less than 50% of the c e l l s w i l l survive. Section 2.3.1. CVL The conventional assay. D/A D i g i t a l to analog conversion. DMF Dose modifying f a c t o r , defined page 10. FCS F e t a l c a l f serum. I/O Input and output. k i l l e d The c l a s s i f i c a t i o n used f o r the low dose assay when the colony formed by an i r r a d i a t e d c e l l a f t e r seven days of incubation has fewer than f i f t y c e l l s . LDA The low dose assay of c e l l s u r v i v a l . LDACLASS ALDAS program used f o r c l a s s i f y i n g c e l l s , section 2.4.4. LDALOC ALDAS program used f o r l o c a t i n g c e l l s , section 2.4.3. LET Linear energy t r a n s f e r . l o c a t i o n day The day on which c e l l s are located when using the low dose assay. This i s generally 2-3 days following i r r a d i a t i o n . low dose assay The c e l l s u r v i v a l assay used i n t h i s study when i t i s estimated that greater than 50% of the c e l l s w i l l survive. Section 2.3.2. LIST OF ABBREVIATIONS AND GLOSSARY OF TERMS (continued) MSCAN ALDAS subroutine used for moving the microscope stage, section 2 . 4 . 2 . PER Oxygen enhancement r a t i o . p ossible e r r o r The c l a s s i f i c a t i o n used i n the low dose assay when a c e l l located on l o c a t i o n day cannot be found (or may have been debris which was mistaken f o r a c e l l ) on c l a s s i f i c a t i o n day. survivor The c l a s s i f i c a t i o n of an i r r a d i a t e d c e l l which can grow into a colony of more than f i f t y c e l l s i n seven days. This c l a s s i f i c a t i o n i s used f o r both the conventional and the low dose assays. TTL T r a n s i s t o r - t r a n s i s t o r l o g i c . A l o g i c a l (electronic) s i g n a l which con s i s t s of a change i n voltage from zero to f i v e V o l t s then back to zero V o l t s . An inverted TTL pulse i s a change i n voltage from f i v e to zero V o l t s then back to f i v e V o l t s . x ACKNOWLEDGEMENTS I g r a t e f u l l y a c k n o w l e d g e t h e f i n i n a n c i a l s u p p o r t I r e c e i v e d i n t h e f o r m o f a two y e a r N a t i o n a l S c i e n c e s and E n g i n e e r i n g R e s e a r c h s c h o l a r s h i p and t h e s u p p o r t p r o v i d e d by t h e B . C . C a n c e r R e s e a r c h F o u n d a t i o n w h i c h p r o v i d e d f o r l a b o r a t o r y m a t e r i a l s and t h e v e r y w o r t h w i l e c o n f e r e n c e and f i e l d t r i p s . I w o u l d l i k e t o e x p r e s s my a p p r e c i a t i o n t o D r . B r a n k o P a l c i c f o r t h e e n e r g y he so f r e e l y s p e n t i n h e l p i n g t o f o r m u l a t e and c a r r y o u t my r e s e a r c h g o a l s , D r . L l o y d S k a r s g a r d f o r h i s s c h o l a r l y c r i t i c i s m s , D r . G e o f f Hof fmann f o r s i t t i n g on my t h e s i s c o m m i t t e e and r e v i e w i n g my t h e s i s , and D r . C o r n e l i u s T o b i a s f o r o f f e r i n g e n c o u r a g e m e n t and i n s i g h t . I c o u l d n e v e r have s u c c e e d e d w i t h o u t t h e warmth and humorous e n c o u r a g e m e n t s o f f e r e d by my f a m i l y , f r i e n d s , and a s s o c i a t e s who h e l p e d make e a c h day memorable i f n o t e n j o y a b l e . I t h a n k a l l t h o s e r e s p o n s i b l e f o r t h e f r e s h c e l l medium, t h e s t e r i l i z e d p i p e t t e s and f l a s k s , t h e c o r n y c o n t a m i n a t i o n j o k e s , t h e l a u g h t e r o v e r s p i l t c u l t u r e s , and e s p e c i a l l y t h e d e p r e s s i o n - r e l i e v i n g c innamon b u n s ! W i t h s p e c i a l c o n s i d e r a t i o n I t h a n k my w i f e f o r h e r l o v e , d e v o t i o n , u n d e r s t a n d i n g , and p e r s e v e r a n c e d u r i n g t h i s f i r s t , w o n d e r f u l n i n e months o f o u r m a r r i e d l i f e and my m o t h e r and f a t h e r who have so l o v i n g l y e n c o u r a g e d me t o t h i s b e g i n n i n g . To t h e s e t h r e e v e r y s p e c i a l p e o p l e I d e d i c a t e t h i s t h e s i s : C y n t h i a G a y l e W i t w i c k i J o h n P e t e r Faddegon A u d r e y May Faddegon x i 1 INTRODUCTION AND HISTORICAL REVIEW 1.1 THE SCIENCE OF RADIOBIQLQGY. D u r i n g t h e l a s t c e n t u r y , t h a n k s t o t h e advancements o f t h e ' a g e o f s c i e n c e ' , p e o p l e have become aware o f t h e e l e c t r o m a g n e t i c and p a r t i c u l a t e r a d i a t i o n s w h i c h a r e a p a r t o f e v e r y d a y l i v i n g . The d i s c o v e r y and s u b s e q u e n t s t u d y o f t h e s e m a n i f e s t a t i o n s o f e n e r g y have r e s u l t e d i n s i g n i f i c a n t c h a n g e s i n t h e l i f e s t y l e s o f most p e o p l e and i n t h e way s c i e n t i s t s v i e w t h e u n i v e r s e I I t i s i m p o r t a n t t o l e a r n how we c a n b e n e f i t f r o m r a d i a t i o n w i t h t h e minimum o f d e t r i m e n t a l e f f e c t s . R a d i o b i o l o g y i s t h e s t u d y o f t h e r e s p o n s e o f b i o l o g i c a l s y s t e m s t o r a d i a t i o n . The m o t i v a t i o n f o r r e s e a r c h i n r a d i o b i o l o g y i s t h r e e f o l d . F i r s t , r a d i a t i o n c o n s t i t u t e s a h e a l t h h a z a r d , and i n t h i s age o f i n c r e a s e d e x p o s u r e i t i s i m p o r t a n t t o e v a l u a t e t h e e f f e c t s o f r a d i a t i o n on a l l f o r m s o f l i f e . S e c o n d , r a d i a t i o n c a n be u s e d as a t o o l f o r m e d i c a l t r e a t m e n t and d i a g n o s i s . T h i s t o o l w i l l be most e f f e c t i v e when t h e e f f e c t o f r a d i a t i o n on l i v i n g s y s t e m s i s w e l l u n d e r s t o o d . T h i r d , t h e r e i s t h e n a t u r a l human i n s t i n c t o f p i o n e e r i n g and d i s c o v e r y t o be f u l f i l l e d - t o c o n t i n u e t o e x p a n d o u r h o r i z o n s . Thus r a d i o b i o l o g y h a s become an i m p o r t a n t f i e l d o f s c i e n c e i n i t s own r i g h t . 1.2 THE INTERACTION OF IONIZING RADIATION WITH C E L L S . P e r h a p s t h e c l a s s o f r a d i a t i o n s w h i c h p o s e t h e g r e a t e s t h e a l t h h a z a r d i s t h a t w h i c h c a r r i e s s u f f i c i e n t e n e r g y t o i o n i z e m o l e c u l e s , and w h i c h c a n c a u s e i r r e v e r s i b l e damage t o c e l l s , t i s s u e s , and a n i m a l s , e v e n r e s u l t i n g i n d e a t h . However , t h e s e r a d i a t i o n s a r e a l s o o f g r e a t u s e i n t h e d i a g n o s i s and t r e a t m e n t o f human d i s e a s e . R a d i a t i o n w h i c h i s c a p a b l e o f i o n i z i n g 2 m o l e c u l e s i s a p p r o p r i a t e l y c a l l e d i o n i z i n g r a d i a t i o n . Such r a d i a t i o n i n c l u d e s X - r a y s and gamma- rays w h i c h a r e h i g h e n e r g y f o r m s o f e l e c t r o m a g n e t i c r a d i a t i o n ( l i g h t ) , as w e l l as p a r t i c l e r a d i a t i o n s u c h as h i g h e n e r g y e l e c t r o n s , p r o t o n s , a l p h a p a r t i c l e s , p i o n s , and so o n . The most d r a m a t i c e f f e c t s o f t h e s e r a d i a t i o n s on b i o l o g i c a l s y s t e m s , s u c h as m u t a t i o n and d e a t h , a r e t h e c o n s e q u e n c e o f t h e f o r m a t i o n o f m o l e c u l a r i o n s w h i c h c a n d i r e c t l y o r i n d i r e c t l y c a u s e damage t o c e l l u l a r b i o m o l e c u l e s . I f t h e DNA o f t h e c e l l i s damaged, t h i s c a n r e s u l t i n m u t a t i o n and ch romosomal a b e r r a t i o n s w h i c h c a n e v e n t u a l l y t r a n s f o r m t h e c e l l o r r e n d e r t h e c e l l u n a b l e t o d i v i d e . Changes i n t h e p h y s i o l o g y and i n t e g r i t y o f t h e c e l l s w h i c h c o n s t i t u t e a t i s s u e o r o r g a n c a n l e a d t o m a l f u n c t i o n o f t h a t o r g a n and u l t i m a t e l y t o s i c k n e s s o r d e a t h o f t h e a n i m a l . Whereas t h e g e n e r a l scheme o f e v e n t s w h i c h f o l l o w t h e i r r a d i a t i o n o f a b i o l o g i c a l s y s t e m i s known, (Bacq and A l e x a n d e r , 1 9 6 1 ; E l k i n d and W h i t m o r e , 1967 ; O k a d a , 1970 ; H a l l , 1 9 7 3 ) , many d e t a i l s have y e t t o be w o r k e d o u t . F o r e x a m p l e , t h e r e i s c u r r e n t l y a g r e a t d e a l o f i n t e r e s t i n t h e mechan isms by w h i c h DNA damage i s i n f l i c t e d and s u b s e q u e n t l y m o d i f i e d b y c e l l u l a r p r o c e s s e s f o l l o w i n g t r e a t m e n t by r a d i a t i o n (Chapman and G i l l e s p i e , 1 9 8 1 ) . I n o r d e r t o s t u d y t h e e f f e c t s o f r a d i a t i o n on b i o l o g i c a l s y s t e m s s c i e n t i s t s have c h o s e n t o s t u d y v a r i o u s b i o l o g i c a l e n d p o i n t s s u c h as DNA s t r a n d b r e a k s , c e l l m u t a t i o n , l o s s o f c e l l p r o l i f e r a t i v e c a p a c i t y , l o s s o f o r g a n f u n c t i o n , d e a t h o f w h o l e a n i m a l s , e t c . The e f f e c t s o f v a r i o u s c o m b i n a t i o n s o f d r u g s , t e m p e r a t u r e , r a d i a t i o n , and o t h e r a g e n t s a r e q u a n t i f i e d i n t e r m s o f t h e i r e f f e c t s on t h e s e e n d p o i n t s . The g o a l i s t o t r y t o e l u c i d a t e t h e e v e n t s t h a t t a k e p l a c e b e t w e e n t h e t r e a t m e n t and t h e o b s e r v e d e f f e c t . 3 1.2.1 Quantifying e f f e c t s with the conventional assay of c e l l  s u r v i v a l . The loss of c e l l p r o l i f e r a t i v e capacity, or c e l l death, has been a widely used endpoint i n radiobiology experiments since i t was f i r s t demonstrated i n mammalian c e l l s by Puck and Marcus i n 1956. Generally, one determines loss of p r o l i f e r a t i v e capacity by p l a t i n g c e l l s exposed to a given treatment such as i o n i z i n g r a d i a t i o n and allowing them to divide f o r about one week under optimal growth conditions. Untreated c e l l s are also plated as a c o n t r o l . Since some c e l l s which divide i n i t i a l l y , eventually lose t h e i r p r o l i f e r a t i v e capacity, an a r b i t r a r y cut o f f i s chosen. Single c e l l s which are able to grow into colonies of f i f t y or more c e l l s during the one week incubation are c a l l e d 'survivors'. A l l other c e l l s are considered to be ' k i l l e d ' . Some marginal surviving colonies may eventually lose t h e i r capacity to divide further and w i l l therefore d i e , while some marginally ' k i l l e d ' colonies may continue to slowly divide and therfore survive. Generally these marginal colonies do not s i g n i f i c a n t l y a f f e c t the r e s u l t s . The l e v e l of c e l l s u r v i v a l f o r a given treatment i s defined as the proportion of treated c e l l s which survive r e l a t i v e to the proportion of untreated c e l l s which survive. In t h i s way the e f f e c t of the treatment can be q u a n t i f i e d i n terms of c e l l s u r v i v a l . This provides a method f or comparison of the effectiveness of d i f f e r e n t treatments. There are many reasons why t h i s endpoint has become popular among r a d i o b i o l o g i s t s : 1. The c e l l s u r v i v a l endpoint i s p a r t i c u l a r i l y u s eful i n studies r e l a t e d to radiotherapy, the treatment of cancer with i o n i z i n g r a d i a t i o n . Ionizing r a d i a t i o n i s used to prevent tumor c e l l s from p r o l i f e r a t i n g , thereby curing the cancer. 2. Experiments performed i n v i t r o are generally easier and l e s s expensive than experiments performed i n  vivo. 3. Observations from c e l l s u r v i v a l experiments can often be 4 e x t r a p o l a t e d t o i n v i v o s t u d i e s and t h e r e b y p r o v i d e m o t i v a t i o n and d i r e c t i o n f o r r e s e a r c h i n v i v o . 4 . T h i s e n d p o i n t c a n a l s o a i d i n u n d e r s t a n d i n g t h e e v e n t s w h i c h e v e n t u a l l y l e a d t o c e l l d e a t h a f t e r i r r a d i a t i o n . I f t h e mechan isms o f t h e e v e n t s a r e s u f f i c i e n t l y u n d e r s t o o d , one s h o u l d be a b l e t o q u a n t i t a t i v e l y p r e d i c t t h e e f f e c t o f a m o d i f i c a t i o n o f t h e t r e a t m e n t o f a c e l l on c e l l s u r v i v a l (and o t h e r e n d p o i n t s ) . I n a d d i t i o n , c e l l s u r v i v a l i s r e l a t i v e l y e a s y t o a s s a y . S e v e r a l v e r y i n t e r e s t i n g b i o l o g i c a l e f f e c t s have b e e n o b s e r v e d u s i n g t h e c e l l s u r v i v a l e n d p o i n t . T h e s e e f f e c t s a r e d i s c u s s e d more c o m p l e t e l y e l s e w h e r e . ( E l k i n d and W h i t m o r e , 1976; O k a d a , 1970; H a l l , 1973). B e c a u s e c e l l s u r v i v a l d e c r e a s e s v e r y r a p i d l y ( a l m o s t e x p o n e n t i a l l y ) w i t h d o s e , s u r v i v a l c u r v e s a r e g e n e r a l l y p l o t t e d on s e m i - l o g p a p e r as i n f i g u r e 1. S u r v i v a l c u r v e s o f mammal ian c e l l s e x p o s e d t o r a d i a t i o n g e n e r a l l y e x h i b i t an i n i t i a l s h o u l d e r , t h o u g h t t o r e p r e s e n t a c c u m u l a t i o n o f s u b l e t h a l damage w h i c h t h e c e l l c a n r e p a i r , f o l l o w e d by a much s t e e p e r , v i r t u a l l y e x p o n e n t i a l d e c r e a s e i n s u r v i v a l w i t h h i g h e r d o s e s . A few i m p o r t a n t c e l l s u r v i v a l e f f e c t s o f r e l e v a n c e t o t h i s t h e s i s a r e d i s c u s s e d h e r e : 1. The s p a t i a l d i s t r i b u t i o n o f i o n i z i n g e v e n t s c a u s e d by t h e i n c i d e n t r a d i a t i o n c a n a f f e c t c e l l s u r v i v a l . T h i s i s g e n e r a l l y e x p l a i n e d a s f o l l o w s : The more damage a c c u m u l a t e d n e a r t h e s e n s i t i v e s i t e o f t h e c e l l , t h e more l i k e l y t h e c e l l w i l l d i e , so t h a t s p a r s e l y i o n i z i n g r a d i a t i o n s a r e n o t as e f f e c t i v e as d e n s e l y i o n i z i n g r a d i a t i o n s . T h i s i s o f t e n r e f l e c t e d i n a r e d u c e d w i d t h o f t h e s h o u l d e r o f t h e s u r v i v a l c u r v e . The l i n e a r e n e r g y t r a n s f e r (LET) o f a g i v e n r a d i a t i o n i s d e f i n e d a s t h e e n e r g y d e p o s i t e d p e r u n i t t r a c k l e n g t h o f t h e p r i m a r y i o n i z i n g p a r t i c l e . The s p a t i a l d i s t r i b u t i o n o f i o n i z i n g e v e n t s f r o m a g i v e n e n e r g y o f r a d i a t i o n i s c h a r a c t e r i z e d by t h e LET o f t h e r a d i a t i o n as w e l l as by t h e d i m e n s i o n s o f 5 2 o r-o < cc C5 > 1.0 0.1 0 . 0 1 > cc 3 0 . 0 0 1 CO T 1 1 I C H O C E L L S S U M M A T I O N ( 1 9 7 9 - 1 9 8 1 ) \ o 2 0 5 10 15 2 0 2 5 3 0 D O S E ( G r a y ) Figure 1: SURVIVAL CURVE SHOWING THE OXYGEN EFFECT. Exponentially growing CHO ce l l s were irradiated in dilute suspensions at 0°C under aerobic (O?)' o r hypoxic {H^) conditions (x-ray source: Picker, 280 Kvpf. Cells were assayed for survival. The ce l l s irradiated under aerobic conditions are much more sensitive than those irradiated under hypoxic conditions, with an OER of about 2.8 at a survival level of 0.01. (Reprinted with permission from Palcic, Brosing, and Skarsgard, 1982). 6 t h e r a d i a t i o n t r a c k , t h e t r a c k c o r e . T h e r e i s a p o i n t f o r v e r y d e n s e l y i o n i z i n g r a d i a t i o n a f t e r w h i c h an i n c r e a s e i n t h e d e n s i t y o f i o n i z a t i o n s w i l l n o t s i g n i f i c a n t l y i n c r e a s e t h e l i k e l i h o o d o f t h e c e l l d y i n g . T h i s i s t h e so c a l l e d ' o v e r k i l l 1 r e g i o n w h i c h o c c u r s f o r r a d i a t i o n s w i t h L E T ' s o f a p p r o x i m a t e l y 200 k e V / u m o r g r e a t e r . I n g e n e r a l , LET i s a c o m p l e x f u n c t i o n o f r a d i a t i o n e n e r g y . (ICRU r e p o r t on L E T , 1 9 7 0 ) . 2 . The s e n s i t i v i t y o f c e l l s t o r a d i a t i o n d e p e n d s on t h e i r l o c a t i o n i n t h e c e l l c y c l e . T h i s c a n be g e n e r a l l y e x p l a i n e d by t h e i m p o r t a n c e o f t h e p r o c e s s o f DNA r e p a i r . M o s t c e l l s d i s p l a y maximum s e n s i t i v i t y t o r a d i a t i o n i m m e d i a t e l y b e f o r e t h e r e p l i c a t i o n s t a g e and d u r i n g m i t o s i s . I f t h e DNA h a s l i t t l e t i m e t o r e p a i r damage b e f o r e r e p l i c a t i n g t h e n i n f o r m a t i o n i n t h e DNA may be l o s t d u r i n g r e p l i c a t i o n . 3 . I n f r a c t i o n a t e d d o s e e x p e r i m e n t s c e l l s a r e i r r a d i a t e d , a l l o w e d t o g row u n d e r o p t i m a l g r o w t h c o n d i t i o n s f o r a w h i l e , t h e n i r r a d i a t e d a g a i n . These e x p e r i m e n t s show t h a t c e l l s a r e more l i k e l y t o s u r v i v e i f t h e r a d i a t i o n i s d e l i v e r e d i n f r a c t i o n s , i n d i c a t i n g t h a t damage i s r e p a i r e d b e t w e e n i r r a d i a t i o n s . C e l l s become l e s s s e n s i t i v e t o i r r a d i a t i o n a f t e r a p e r i o d o f i n c u b a t i o n l o n g enough f o r t h e r e p a i r p r o c e s s e s t o t a k e p l a c e . ( E l k i n d and S u t t o n , 1 9 5 9 ) . T h i s e v i d e n c e i s s u p p o r t e d by dose r a t e e x p e r i m e n t s w h i c h show t h a t c e l l s a r e more s e n s i t i v e t o h i g h e r dose r a t e s . 4 . C e l l s c o n t a i n i n g o x y g e n ( o x i c c e l l s ) a r e g e n e r a l l y more s e n s i t i v e t h a n c e l l s c o n t a i n i n g l i t t l e o x y g e n ( h y p o x i c c e l l s ) . T h i s e f f e c t , t h e so c a l l e d ' o x y g e n e f f e c t 1 , i s i m p o r t a n t enough t h a t a s p e c i a l t e r m was d e f i n e d t o q u a n t i f y i t : t h e o x y g e n enhancement r a t i o (OER). OER i s d e f i n e d as t h e r a t i o o f t h e dose w i t h o u t o x y g e n t o t h e dose w i t h o x y g e n t o y i e l d t h e same s u r v i v a l l e v e l . F o r mammalian c e l l s t h i s r a t i o i s a p p r o x i m a t e l y 3 . 0 a t a s u r v i v a l l e v e l o f 0 . 0 1 . ( F i g u r e 1 , f r o m P a l c i c e t a l , 1982 . ) T h u s , i n t h e 7 p r e s e n c e o f o x y g e n , a c e l l needs o n l y one t h i r d o f t h e dose i t r e q u i r e s i n t h e a b s e n c e o f o x y g e n t o r e s u l t i n n i n e t y - n i n e o u t o f one h u n d r e d c e l l s b e i n g k i l l e d a s a c o n s e q u e n c e o f i r r a d i a t i o n ! A g e n t s s u c h a s o x y g e n w h i c h i n t e r a c t w i t h r a d i a t i o n t o d e c r e a s e c e l l s u r v i v a l a r e c a l l e d r a d i o s e n s i t i z e r s . 1 . 2 . 2 Measurements o f c e l l s u r v i v a l a t l o w d o s e s o f i o n i z i n g  r a d i a t i o n . M o s t o f t h e s u r v i v a l d a t a i n t h e l i t e r a t u r e m e a s u r e s s u r v i v a l l e v e l s o f a b o u t 0 . 5 o r l e s s . T h e r e i s now ample m o t i v a t i o n f o r m e a s u r i n g s u r v i v a l s a t h i g h e r s u r v i v a l l e v e l s : 1 . F o r d e c a d e s t h e r e s u l t s o f i n  v i t r o s t u d i e s on c e l l s h a v e b e e n e x t r a p o l a t e d t o c e l l s i n v i v o . Some o f t h e s e r e s u l t s a r e a p p l i e d t o r a d i o t h e r a p y . G e n e r a l l y t h e t r e a t m e n t o f c a n c e r w i t h r a d i a t i o n i n v o l v e s d a i l y d o s e s o f 1 .0 t o 3 . 0 Gy ( f o r t r e a t m e n t w i t h c u r a t i v e i n t e n t , p a l l i a t i v e d o s e s a r e somet imes h i g h e r ) f o r p e r i o d s o f two t o s i x w e e k s . L a b o r a t o r y s t u d i e s o f c e l l i n a c t i v i a t i o n , on t h e o t h e r h a n d , n o r m a l l y u s e d o s e s o f 5 - 3 0 Gy i n o r d e r t o p r o d u c e e f f e c t s l a r g e enough t o measure a c c u r a t e l y . T h u s , t h e d o s e s o f r a d i a t i o n u s e d i n r a d i o t h e r a p y (pe r f r a c t i o n ) a r e as much as an o r d e r o f m a g n i t u d e l o w e r t h a n t h o s e d o s e s u s e d f o r t h e i n v i t r o s t u d i e s o f c e l l s u r v i v a l . Measurements a t l o w d o s e s o f r a d i a t i o n ( h i g h c e l l s u r v i v a l ) must be made t o c o n f i r m t h a t t h e e f f e c t s o b s e r v e d a t l o w s u r v i v a l l e v e l s a r e a l s o o b s e r v e d a t h i g h s u r v i v a l l e v e l s . 2 . C e r t a i n b a s i c c o n c e p t s o f r a d i o b i o l o g y s u c h a s t h e n a t u r e o f t h e c r i t i c a l l e s i o n i n DNA and t h e d e g r e e o f i m p o r t a n c e o f DNA r e p a i r i n c e l l d e a t h a r e s t i l l c o n t r o v e r s i a l . M o d e l s b a s e d on s u c h c o n c e p t s make v a r i o u s p r e d i c t i o n s o f t h e s u r v i v a l o f c e l l s a t l o w d o s e s o f r a d i a t i o n . Low dose measurements o f c e l l s u r v i v a l may h e l p t o d i s t i n g u i s h 8 b e t w e e n t h e s e m o d e l s and t o d e t e r m i n e t h e r e l a t i v e i m p o r t a n c e o f t h e i r u n d e r l y i n g a s s u m p t i o n s . Many f e e l t h a t t h e r e s p o n s e o f c e l l s t o t h e f i r s t few g r y s c o n s t i t u t e t h e most i m p o r t a n t a s p e c t o f c e l l k i l l i n g ( e g . , A l p e r , 1 9 7 9 ) . The s i x t h L . H . G r a y c o n f e r e n c e i n London i n 1974 was d e v o t e d t o c e l l s u r v i v a l a t l o w d o s e s . ( A l p e r , e d , 1974) I f measurement o f c e l l s u r v i v a l a t l o w d o s e s i s i m p o r t a n t , why i s t h e r e so l i t t l e d a t a a v a i l a b l e ? P e r h a p s t h e m a j o r r e a s o n i s t h e l a r g e u n c e r t a i n t y i n h e r e n t i n c e l l s u r v i v a l d a t a . I f a c c u r a t e measurements a r e t o be made u s i n g c o n v e n t i o n a l p r o c e d u r e s , i n o r d e r t o c o l l e c t s u f f i c i e n t d a t a f o r r e a s o n a b l e s t a t i s t i c s t h e e x p e r i m e n t s w o u l d be v e r y t i m e c o n s u m i n g . S i n c e t i m e i s a c r u c i a l f a c t o r i n s u r v i v a l e x p e r i m e n t s , t h e r e s u l t s o f s u c h l e n g t h y e x p e r i m e n t s w o u l d be i n j e o p a r d y . E v e n i f e x p e r i m e n t s a r e p e r f o r m e d p e r f e c t l y w i t h no s i g n i f i c a n t e x p e r i m e n t e r e r r o r s and minimum measurement u n c e r t a i n t i e s t h e r e a r e two m a j o r s o u r c e s o f e r r o r i n t h e c o n v e n t i o n a l a s s a y o f c e l l s u r v i v a l . T h e s e a r e t h e s t a t i s t i c a l v a r i a t i o n s i n t h e number o f c e l l s p l a t e d as e s t i m a t e d f r o m t h e vo lume o f t h e sample p l a t e d f r o m an a c c u r a t e l y measu red c o n c e n t r a t i o n ,of c e l l s and t h e s t a t i s t i c a l v a r i a t i o n i n t h e p r o p o r t i o n o f c e l l s w i t h i n t h i s p l a t e d samp le vo lume t h a t a r e ' d e s t i n e d ' t o s u r v i v e . I n a w e l l m i x e d samp le b o t h t h e p l a t e d c e l l s and t h e s u r v i v i n g p l a t e d c e l l s have a P o i s s o n d i s t r i b u t i o n . The s e c o n d u n c e r t a i n t y c a n o n l y be r e d u c e d by p l a t i n g a l a r g e number o f c e l l s . The f i r s t u n c e r t a i n t y c a n be v i r t u a l l y e l i m i n a t e d by a c c u r a t e l y c o u n t i n g t h e number o f c e l l s p l a t e d , i n s t e a d o f e s t i m a t i n g t h i s number . I n p r a c t i c e t h i s w i l l a l s o s i g n i f i c a n t l y i n c r e a s e t h e p l a t i n g e f f i c i e n c y o f t h e e x p e r i m e n t , t h e r e l a t i v e number o f u n t r e a t e d 9 c e l l s t h a t s u r v i v e , s i n c e a u t o m a t i c c e l l c o u n t e r s g e n e r a l l y c o u n t d e b r i s a l o n g w i t h c e l l s r e s u l t i n g i n an o v e r e s t i m a t e o f t h e c o n c e n t r a t i o n o f c e l l s i n a s a m p l e . An i n c r e a s e d p l a t i n g e f f i c i e n c y w i l l f u r t h e r d e c r e a s e t h e u n c e r t a i n t y i n t h e m e a s u r e d s u r v i v a l . The l o w d o s e a s s a y , as i t i s r e f e r r e d t o i n t h i s t h e s i s , makes u s e o f a m i c r o s c o p e f o r l o c a t i n g p l a t e d c e l l s t o be a s s a y e d f o r s u r v i v a l . T h i s a s s a y w o u l d be v e r y t i m e c o n s u m i n g and e x t r e m e l y t i r i n g were t h e c e l l l o c a t i o n s t o be p e r f o r m e d c o m p l e t e l y m a n u a l l y , and t h e g a i n i n a c c u r a c y o b t a i n e d by l o c a t i n g c e l l s w o u l d be l o s t s i n c e c o m p a r i t i v e l y few c e l l s c o u l d be l o c a t e d i n a r e a s o n a b l e t i m e . Thus a t l e a s t some d e g r e e o f a u t o m a t i o n i s r e q u i r e d i n o r d e r t o make t h i s p r o c e d u r e a v i a b l e a l t e r n a t i v e t o t h e c o n v e n t i o n a l a s s a y f o r m e a s u r i n g h i g h s u r v i v a l l e v e l s . I n f a c t i t i s p o s s i b l e t o a u t o m a t e t h e a s s a y t o s u c h an e x t e n t t h a t a compute r c o u l d do most o f t h e work v e r y q u i c k l y . T h i s a s s a y s i g n i f i c a n t l y r e d u c e s t h e u n c e r t a i n t y i n t h e number o f c e l l s p l a t e d . I n a d d i t i o n , t h e number o f s u r v i v i n g c e l l s p l a t e d h a s a b i n o m i a l d i s t r i b u t i o n s i n c e t h e t o t a l number o f c e l l s p l a t e d i s a c c u r a t e l y known. T h i s f u r t h e r i n c r e a s e s t h e a c c u r a c y o f t h i s a s s a y . These u n c e r t a i n t i e s a r e d i s c u s s e d more c o m p l e t e l y i n s e c t i o n 4 . 1 . 1 . 2 . 3 Case i n p o i n t f o r l o w d o s e s t u d i e s : t h e o x y g e n e f f e c t . S t u d y o f t h e o x y g e n e f f e c t has added t o o u r k n o w l e d g e o f r a d i o b i o l o g y and h a s h e l p e d t o s o r t o u t t h e mechan isms i n v o l v e d i n t h e e v e n t s w h i c h l e a d t o c e l l d e a t h . The o x y g e n e f f e c t may have i m p o r t a n t c o n s e q u e n c e s f o r r a d i o t h e r a p y . One p o s s i b l e c o m p l i c a t i o n i n t h e t r e a t m e n t o f t u m o r s i s t h a t t h e y c o n t a i n r e g i o n s o f h y p o x i c c e l l s . When a tumor i s t r e a t e d w i t h i o n i z i n g r a d i a t i o n t h e r e i s c o n c e r n t h a t a l t h o u g h t h e w e l l o x y g e n a t e d c e l l s a r e i n a c t i v a t e d ( c a n n o t p r o l i f e r a t e ) , t h e r a d i a t i o n w i l l have l e s s e f f e c t on t h e h y p o x i c 10 c e l l s , and t h e s u r v i v o r s w i l l p r o l i f e r a t e upon r e o x y g e n a t i o n , e v e n t u a l l y r e s u l t i n g i n r e g r o w t h o f t h e t u m o r . ( T h o m l i n s o n and G r a y , 1 9 5 5 ) . S i n c e h y p o x i c c e l l s a r e a p r o b l e m i n tumor t r e a t m e n t i t w o u l d be v a l u a b l e t o s t u d y r a d i o s e n s i t i z e r s w h i c h c a n m i m i c t h e e f f e c t o f o x y g e n i n s e n s i t i z i n g h y p o x i c c e l l s t o i o n i z i n g r a d i a t i o n . (Adams, 1 9 7 3 ) . N i t r o i m i d a z o l e s a r e s u c h a c l a s s o f d r u g s . (Wardman, 1 9 7 7 ) . They a r e b e l i e v e d t o s e n s i t i z e h y p o x i c c e l l s t o r a d i a t i o n as a r e s u l t o f t h e i r h i g h e l e c t r o n a f f i n i t y . (Oxygen a l s o h a s a h i g h e l e c t r o n a f f i n i t y . ) The n i t r o i m i d a z o l e s do n o t i n c r e a s e t h e s e n s i t i v i t y o f w e l l o x y g e n a t e d c e l l s t o r a d i a t i o n . ( e . g . Wardman, 1977 . ) The d e g r e e o f r a d i o s e n s i t i z a t i o n b y t h e s e d r u g s c a n be e x p r e s s e d i n t e r m s o f t h e i r d o s e m o d i f y i n g f a c t o r (DMF). DMF i s d e f i n e d as t h e r a t i o o f t h e d o s e w i t h o u t t h e d r u g t o t h e dose i n t h e p r e s e n c e o f t h e d r u g t o y i e l d t h e same s u r v i v a l l e v e l . T h i s i s a n a l a g o u s t o t h e d e f i n i t i o n o f OER. M e t r o n i d a z o l e was one o f t h e f i r s t n i t r o i m i d a z o l e s t o be s t u d i e d i n d e t a i l . I t has b e e n shown t o be a r a d i o s e n s i t i z e r o f h y p o x i c mammal ian c e l l s w i t h a maximum DMF o f a b o u t 2 . 0 a t 10 mM c o n c e n t r a t i o n , h a s no m e a s u r a b l e e f f e c t i n o x i c c e l l s , and i s t o x i c t o hypox i c c e l l s . ( F o s t e r and W i l l s o n , 1973 ; A s g u i t h e t a l , 1 9 7 4 ) . M e t r o n i d a z o l e h a s a l s o been shown t o a c t as a r a d i o s e n s i t i z e r i n v i v o . (Begg e t a l , 1974 ; Denekamp e t a l , 1974 and 1 9 7 5 ) . C u r r e n t l y , more a t t e n t i o n i s g i v e n t o m i s o n i d a z o l e w h i c h i s a b e t t e r r a d i o s e n s i t e r a t l o w e r c o n c e n t r a t i o n s h a v i n g a DMF o f a b o u t 2 . 3 a t 15 mM c o n c e n t r a t i o n . (Moore e t a l , 1976 ; M c N a l l y , 1976 ; Adams e t a l , 1 9 7 6 ) . I t h a s a l s o b e e n shown t o a c t as a r a d i o s e n s i t i z e r in v i v o . (Denekamp e t a l , 1974 and 1975 ; T u r n e r e t a l , 1 9 8 0 ) . M i s o n i d a z o l e and a few r e l a t e d compounds a r e a l r e a d y u n d e r g o i n g c l i n i c a l t r i a l s a t a number o f i n s t i t u t i o n s , i n c l u d i n g t h e A . M . Evans 11 C l i n i c i n V a n c o u v e r , B r i t i s h Co lunub ia . Many s t u d i e s show t h a t m i s o n i d a z o l e a p p e a r s t o r a d i o s e n s i t i z e h y p o x i c t u m o r s i n humans. ( D i s c h e , 1978 ; U r t a s u n e t a l , 1977 ; T h o m l i n s o n e t a l , 1 9 7 6 ) . A good r e v i e w o f t h i s s u b j e c t i s g i v e n i n t h e p r o c e e d i n g s o f t h e Key B i s c a y n e C o n f e r e n c e " R a d i a t i o n S e n s i t i z e r s " , (L.W. B r a d y , e d . , 1 9 8 0 ) . T h e r e i s some c o n t r o v e r s y as t o w h e t h e r t h e t r e a t m e n t o f h y p o x i c r e g i o n s i n t u m o r s c a n be i m p r o v e d w i t h r a d i o s e n s i t i z e r s . I n d e e d , r e c e n t f i n d i n g s i n v i t r o i n d i c a t e t h a t o x y g e n i t s e l f h a s l i m i t e d r a d i o s e n s i t i z i n g a b i l i t y a t c l i n i c a l l y u s e f u l d o s e s o f r a d i a t i o n (o f t h e o r d e r o f two Gray ) a l t h o u g h t h e d e g r e e o f s e n s i t i z a t i o n a t t h e s e d o s e s i s d i f f i c u l t t o measure w i t h c u r r e n t t e c h n i q u e s . ( P a l c i c e t a l , 1982 . ) F i g u r e 2 shows t h e measu red e f f e c t o f o x y g e n on C h i n e s e h a m s t e r c e l l s u r v i v a l as m e a s u r e d by P a l c i c e t a l . F o r t h e e x p e r i m e n t a l s y s t e m u s e d i n t h e s e s t u d i e s t h e OER o f o x y g e n i s m e a s u r e d t o be a b o u t 1.5±0.7 f o r s u r v i v a l l e v e l s o f a b o u t 0 . 8 o r g r e a t e r , c o r r e s p o n d i n g t o d o s e s t o o x i c c e l l s o f l e s s t h a n one G r a y . I f o x y g e n i s a p o o r r a d i o s e n s i t i z e r a t l o w d o s e s , a r e t h e n i t r o i m i d a z o l e s w h i c h m i m i c t h e r a d i o s e n s i t i z i n g e f f e c t o f o x y g e n a l s o p o o r r a d i o s e n s i t i z e r s a t l o w d o s e s ? F r a c t i o n a t e d d o s e s t u d i e s have a l r e a d y i n d i c a t e d t h a t m i s o n i d a z o l e may be a p o o r r a d i o s e n s i t i z e r i n v i v o a t l o w d o s e s . (Durand and B r o w n , 1 9 8 0 ) . What i s i t s r a d i o s e n s i t i z i n g a b i l i t y i n v i t r o a t l o w d o s e s ? T h i s s t u d y m e a s u r e s t h e r a d i o s e n s i t i z i n g e f f e c t o f m i s o n i d a z o l e on t h e s u r v i v a l o f C h i n e s e h a m s t e r o v a r y c e l l s a t d o s e s o f r a d i a t i o n as l o w as one G r a y . I t was f o u n d t h a t t h e r a d i o - s e n s i t i z a t i o n p r o d u c e d by m i s o n i d a z o l e , l i k e o x y g e n , d i m i n i s h e s a t l o w r a d i a t i o n d o s e . I f t h i s o b s e r v a t i o n i s r e p r e s e n t a t i v e o f t h e c l i n i c a l s i t u a t i o n , i t may e x p l a i n t h e m i n i m a l s u c c e s s o f m i s o n i d a z o l e i n p a t i e n t t r e a t m e n t s t o d a t e ( e . g . D i s c h e , 1 9 8 0 ) . 12 0.4 0.6 0.8 1 6 8 10 DOSE (Gy) IN 0 2 Figure 2: OXYGEN ENHANCEMENT RATIO AT LOW DOSES OF RADIATION. Exponentially growing CHO cel l s were irradiated in dilute suspensions at 0°C under aerobic or hypoxic conditions. Solid c i r c l e s represent the OER calculated from CHO c e l l survival data obtained at low doses of x-rays. The open c i r c l e i s the OER as calculated from the data of figure 1. The OER i s clearly dose dependent for this experimental system. (Reprinted from Palcic, Brosing, and Skarsgard, 1982 with permission.) 13 MATERIALS AND METHODS 2 . 1 CELL CULTURE PROCEDURES. The i n v i t r o b i o l o g i c a l e x p e r i m e n t s r e p o r t e d h e r e were p e r f o r m e d on C h i n e s e h a m s t e r c e l l l i n e s grown i n t i s s u e c u l t u r e . The C h i n e s e h a m s t e r o v a r y (CHO) c e l l l i n e i s w i d e l y u s e d : i t g rows r a p i d l y , c a n be c l o n e d w i t h h i g h p l a t i n g e f f i c i e n c y , and c a n be grown i n m o n o l a y e r , s u s p e n s i o n , and s o f t a g a r t i s s u e (Thompson, i n J a k o b y and P a s t r i n a , e d . , 1 9 7 9 ) . We grew CHO c e l l s i n s u s p e n s i o n c u l t u r e i n a l p h a medium, s u p p l e m e n t e d w i t h 10% f e t a l c a l f serum ( F C S ) , a n t i b i o t i c s , and b i c a r b o n a t e b u f f e r . The c u l t u r e s were grown i n a s p i n n e r f l a s k , shown i n f i g u r e 3 , i n an i n c u b a t o r a t 37°C; pH i s r e g u l a t e d t o 7 . 4 b y c o n t i n u o u s g a s s i n g w i t h 5% CO^ i n a i r . The c e l l 4 c u l t u r e i s d i l u t e d d a i l y t o a b o u t 7X10 c e l l s / m l , m a i n t a i n i n g a s y n c h r o n o u s e x p o n e n t i a l g r o w t h w i t h a d o u b l i n g t i m e o f a b o u t 12 h o u r s . 2 . 2 IRRADIATION PROCEDURES. The s o u r c e o f r a d i a t i o n u s e d was a P i c k e r X - r a y m a c h i n e , 270 K V P , HVL=1.7 mm C u , a t a dose r a t e o f b e t w e e n 0 . 3 t o 2 . 0 G r a y p e r m i n u t e . The f o l l o w i n g a p p r o a c h , ( P a r k e r e t a l , 1969 ; Thomson and R a u t h , 1 9 7 4 ) , was u s e d f o r i r r a d i a t i n g t h e c e l l s : C e l l s f r o m c u l t u r e were removed t o t h e i r r a d i a t i o n v e s s e l , shown i n f i g u r e 3 , i n d i l u t e s u s p e n s i o n o f a p p r o x i m a t e l y 1 . 2 x 1 0 ^ c e l l s / m l . The v e s s e l was p l a c e d on a wooden p l a t f o r m a t one o f two d i s t a n c e s above t h e head o f t h e X - r a y t u b e o r d i r e c t l y on t h e head and t h e c e l l s were i r r a d i a t e d . The h e i g h t o f t h e v e s s e l above t h e X - r a y h e a d and t h e t i m e t a k e n f o r i r r a d i a t i o n d e t e r m i n e d t h e d o s e d e l i v e r e d t o t h e c e l l s . The i r r a d i a t i o n s e t - u p i s shown i n f i g u r e 4 . The measurement o f t h e dose r a t e i s d i s c u s s e d i n s e c t i o n 4 . 1 . 2 . S T I R B A R I G a s F I o w Figure 3: THE FLASKS USED FOR GROWING AND IRRADIATING CELLS. Cells are grown in suspension culture in alpha medium supplemented with 10% FCS, antibiotics, and bicarbonate buffer. Cultures are incubated at 37°C in spinner flasks (a); pH i s regulated to 7.4 by continuous gassing with 5% CO^ in a i r . Cells are irradiated in irradiation vessels (b) with continuous^ nitrogen flow over the stirred 15-20 ml suspension of 1.2 x 10 cells/ml. Samples were obtained by removing the outlet stopper b r i e f l y and lowering a pipet down the neck of the vessel into the suspension. A l l experiments were performed at O'C. 15 G A S O U T plexiglass container ice water C E L L S U S P E N S I O N when x = 0.0 cm X dose rate =1.6 Gy/min when x = 10.0 cm dose rate =0.8 Gy/min when x = 49.0 cm dose rate = 0.3 Gy/min X-ray head 20 X 20 era colimator Figure 4: DIAGRAM OF THE IRRADIATION SET-UP. The irradiation vessel (figure 3) was placed i n a plexiglass ice water bath. A s t i r motor was suspended above the vessel. To obtain a dose -rate of 1.6 Gy/min the plexiglass container was placed directly on top of the 20 x 20 cm colimator. To achieve a lower dose rate of 0.8 and 0.3 Gy/min the plexiglass container was supported around the edges by a wooden platform 10 cm and 49 cm above the colimator respectivelyi 16 2 . 3 CELL SURVIVAL ASSAYS. The i r r a d i a t e d s a m p l e s were a s s a y e d f o r c e l l s u r v i v a l by one o f two m e t h o d s : t h e c o n v e n t i o n a l a s s a y o r t h e l o w d o s e a s s a y . These a s s a y s a r e d i a g r a m a t i c a l l y r e p r e s e n t e d i n f i g u r e 5 . 2 . 3 . 1 The c o n v e n t i o n a l a s s a y . C e l l s w i t h e s t i m a t e d s u r v i v a l o f l e s s t h a n 0 . 5 were a s s a y e d f o r c e l l s u r v i v a l u s i n g t h e c o n v e n t i o n a l ' P u c k p l a t i n g ' a s s a y . (Puck and M a r c u s , 1 9 5 6 ) . I m m e d i a t e l y a f t e r i r r a d i a t i o n t h e samp le o f c e l l s was c e n t r i f u g e d t o remove t h e m i s o n i d a z o l e . T h i s i s done b e c a u s e m i s o n i d a z o l e h a s b e e n shown t o be t o x i c t o c e l l s grown u n d e r g r o w t h c o n d i t i o n s and f u r t h e r m o r e , t h i s t o x i c i t y h a s b e e n shown t o i n t e r a c t w i t h t h e damage c a u s e d by i r r a d i a t i o n . (Moore e t a l , 1976 ; S t r a t f o r d and Adams, 1977 ; P a l c i c e t a l , 1978 ; K o r b e l i k e t a l , 1 9 8 1 ) . The c o n c e n t r a t i o n o f c e l l s i n e a c h samp le t o be a s s a y e d f o r s u r v i v a l i s e s t i m a t e d u s i n g t h e C o u l t e r E l e c t r o n i c s c e l l c o u n t e r . A vo lume o f e a c h sample e s t i m a t e d t o c o n t a i n enough c e l l s t o g i v e 1 0 0 - 2 0 0 c o l o n i e s o f 50 c e l l s o r more a r e p l a t e d i n 5 cm d i a m e t e r p e t r i s d i s h e s i n 5ml o f a l p h a medium w i t h 10% F C S , a n t i b i o t i c s , and b i c a r b o n a t e b u f f e r , and grown a t 37°C i n an i n c u b a t o r w i t h 5% C 0 2 f l o w f o r 7 d a y s , a t w h i c h t i m e t h e medium i s d i s c a r d e d and t h e c e l l s a r e s t a i n e d w i t h m e t h y l e n e b l u e . C o l o n i e s o f more t h a n 50 c e l l s p e r c o l o n y a r e d e f i n e d as ' s u r v i v o r s ' , and o f l e s s t h a n 50 c e l l s p e r c o l o n y a r e j u d g e d t o have l o s t t h e i r p r o l i f e r a t i v e c a p a c i t y and a r e d e f i n e d as ' n o n - s u r v i v o r s ' o r ' k i l l e d ' . S u r v i v a l i s e s t i m a t e d as f o l l o w s : S u r v i v a l , S = C _ E q u a t i o n 1 NP C=number o f s u r v i v o r s ( c o l o n i e s ) N=number o f c e l l s p l a t e d P=C/N a t z e r o r a d i a t i o n d o s e ( p l a t i n g e f f i c i e n c y ) 17 TH£ CONVENTIONAL ASSAY TH£ LOU POS£ f\SSt\Y Ceils are ,ce.n. iri fu< rt.stispe.nJ.eJL. are. irra J-io.'teJ.. 0000006% OOOOOOO OOOOOOO OOOOOOO OOOOOOO comptftcr scan, reference . point. /Ipproximateiu 20o/S ceU,s are eC-a.te.cL 2V ecich or thrte pevris dUsheSjuhe, SiS the. estimated. Sarvl h tricretcst pCo."te. uoih. 1G> O-'rmttJcU.s boliont vietJ, 2.Sc*J-fUsK. 'survival*. 300-600 cells are plated, per vessel. Centri.Tu.qpjcIon. is not required, since. ceCLs are HiLated. 2ooo~£x>oo times. cms mero-hle. araJLaa&e. '£ud.e«t Joystick -to t*K.troL s ia*e motion. Cells a-re. Cocojt&cL tolih a. Microscope. #u.tot«a£io/l uitLL eventILO-LLLI replace. tne.jra.da.ate. stu-dentr. Ce,U oUvide S are tncubaieoL for six to ci^ki dUas after pLo-tina^. Cetls cakt'ek. After medium it oiipco.rdedjee.tts weerh trie re. £Man fO ct-CCs. are couttieeL. microscope £,to.g& CeXL locations, are, revisitedo-nol c.t'o.ssCfieoL /Ka/maUu (tcdCou%t)or bit COM.outre\r. c*tojties can. be. /fLoXttoreci on. <x-- /?~:W. ' Figure 5: COMPARISON OF THE CELL SURVIVAL ASSAYS. Two different procedures were used for assaying ce l l s for loss of proliferative a b i l i t y , the c e l l survival assays. The conventional assay i s used for ce l l s estimated to have a survival of less than 50% . The low dose assay i s used for ce l l s with survival of greater than 50%. 18 2 . 3 . 2 The l o w d o s e a s s a y . C e l l s w i t h e s t i m a t e d s u r v i v a l o f g r e a t e r t h a n 0 . 5 were a s s a y e d f o r c e l l s u r v i v a l u s i n g t h e ' l o w dose a s s a y ' . I r r a d i a t e d s a m p l e s were n o t c e n t r i f u g e d s i n c e s a m p l e s were d i l u t e d 2 0 0 0 - 5 0 0 0 f o l d p r i o r t o p l a t i n g , r e d u c i n g t h e c o n c e n t r a t i o n o f m i s o n i d a z o l e t o a l e v e l w h i c h i s n o t t o x i c . ( K o r b e l i k e t a l , 1981) . An a l i q u o t o f 1 0 0 - 4 0 0 c e l l s i s p l a t e d i n a s u i t a b l e v e s s e l f o r v i e w i n g u n d e r t h e m i c r o s c o p e . S u f f i c i e n t medium must be added t o t h e v e s s e l t o r e d u c e m o t i o n o f t h e medium t o p r e v e n t c e l l s f r o m w a n d e r i n g f r o m t h e i r r e s p e c t i v e c o l o n i e s . I f m i c r o t e s t p l a t e s a r e u s e d ( v e s s e l c o n t a i n i n g an 8x12 a r r a y o f 7 mm d i a m e t e r , 10 mm deep w e l l s as shown i n f i g u r e 5) o n l y 200 u l o f medium a r e r e q u i r e d p e r w e l l (20 m l p e r m i c r o t e s t p l a t e ) t o p r e v e n t p r o b l e m s c a u s e d by w a n d e r e r s . I f 25 c m 2 c u l t u r e f l a s k s a r e u s e d ( f i g u r e 5) t h e e n t i r e f l a s k must be f i l l e d w i t h medium (about 50 ml ) t o e l i m i n a t e w a n d e r e r s . W i t h t h e a i d o f a m i c r o s c o p e , c e l l s p l a t e d i n t h e v e s s e l s a r e c o o r d i n a t e l o c a t e d and t h e i r l o c a t i o n s r e c o r d e d two o r t h r e e d a y s a f t e r e x p o s u r e t o r a d i a t i o n . S i x t o e i g h t d a y s f o l l o w i n g i r r a d i a t i o n e a c h c o o r d i n a t e l o c a t i o n i s r e - e x a m i n e d . C o l o n i e s w i t h g r e a t e r t h a n 50 c e l l s a r e d e f i n e d as ' s u r v i v o r s ' and c o l o n i e s w i t h l e s s t h a n 50 c e l l s a r e d e f i n e d a s ' k i l l e d ' . On l o c a t i o n d a y , o n l y t h o s e l o c a t i o n s c o n s i d e r e d t o r e p r e s e n t p o t e n t i a l l y v i a b l e c e l l s a r e r e c o r d e d ; t h a t i s , l y s e d c e l l s and c e l l d e b r i s a r e n o t r e c o r d e d . T h i s i s s u b j e c t t o a c e r t a i n d e g r e e o f e x p e r i m e n t e r b i a s w h i c h i s t a k e n i n t o a c c o u n t as f o l l o w s : On t h e c l a s s i f i c a t i o n day l o c a t i o n s w h i c h have no v i a b l e c e l l s a r e c l a s s i f i e d as ' p o s s i b l e e r r o r ' and c o n s t i t u t e an e x p e r i m e n t a l u n c e r t a i n t y . On c l a s s i f i c a t i o n day t h e o b s e r v e r r e t u r n s t o t h e l o c a t i o n s r e c o r d e d on t h e l o c a t i o n day and c l a s s i f i e s t h e 19 c o l o n i e s as ' s u r v i v o r s ' , ' k i l l e d ' , ' c o i n c i d e n t ' w i t h a n o t h e r c o l o n y , o r ' p o s s i b l e e r r o r ' . Two c o l o n i e s a r e c o n s i d e r e d c o i n c i d e n t i f t h e c e n t r e o f e i t h e r one o f t h e c o l o n i e s i s w i t h i n 100 um (about 5 - 1 0 c e l l d i a m e t e r s ) o f t h e p e r i m e t e r o f t h e n e i g h b o u r i n g c o l o n y . T h i s d e f i n i t i o n i s u s e d t o p r e v e n t f a v o u r i n g s u r v i v i n g c o l o n i e s o v e r c o l o n i e s o f a few c e l l s . C o i n c i d e n c e s were n o t i n c l u d e d i n t h e c a l c u l a t i o n o f c e l l s u r v i v a l . ( I f two n e a r b y c e l l s p r o l i f e r a t e , t h e s e p a r a t e c o l o n i e s c a n g e n e r a l l y be d i s t i n g u i s h e d e v e n t h o u g h t h e y o v e r l a p . I f e i t h e r c o l o n y w o u l d have grown o v e r i t s n e i g h b o u r i f t h e n e i g h b o u r was n o n - p r o l i f e r a t i n g , t h e c o l o n i e s s h o u l d be c o n s i d e r e d c o i n c i d e n t . O t h e r w i s e t h e r e l a t i v e p r o p o r t i o n o f s u r v i v i n g c e l l s r e c o r d e d as c o i n c i d e n t w i l l g e n e r a l l y be l e s s t h a n t h e r e l a t i v e p r o p o r t i o n o f n o n - s u r v i v i n g c e l l s t h a t a r e r e c o r d e d as c o i n c i d e n t . I t i s d i f f i c u l t t o i d e n t i f y a c o l o n y o f o n l y a few c e l l s i f i t i s w i t h i n a b o u t 100 um o f a n o t h e r c o l o n y . Thus a 100 um ' r a d i u s o f c o i n c i d e n c e was e m p l o y e d . ) T h e r e was a s i g n i f i c a n t number o f c o i n c i d e n c e s i n m i c r o t e s t p l a t e s i f more t h a n a b o u t 200 c e l l s were p l a t e d p e r p l a t e ( a v e r a g e o f two c e l l s p e r w e l l ) . A f t e r t h e l o w d o s e a s s a y was a u t o m a t e d and c e l l s w e r e p l a t e d i n 25 c m 2 f l a s k s c o i n c i d e n c e s were e a s i l y a v o i d e d . I f a c o l o n y c o u l d n o t be f o u n d a t t h e r e c o r d e d l o c a t i o n on c l a s s i f i c a t i o n day o r i f t h e r e were no v i a b l e c e l l s i n t h a t c o l o n y t h e l o c a t i o n i s c l a s s i f i e d as ' p o s s i b l e e r r o r ' . H a l f o f t h e ' p o s s i b l e e r r o r ' c o l o n i e s a r e c o u n t e d as dead c o l o n i e s , t h e o t h e r h a l f a r e c o n s i d e r e d l o c a t i o n e r r o r s . The ' p o s s i b l e e r r o r ' c l a s s i f i c a t i o n s r e s u l t i n an u n c e r t a i n t y i n t h e number o f c e l l s p l a t e d ( e q u a t i o n 2) w h i c h i s e s t i m a t e d a s h a l f o f t h e ' p o s s i b l e e r r o r 1 c o l o n i e s . The number o f ' p o s s i b l e e r r o r ' c o l o n i e s was g e n e r a l l y s i g n i f i c a n t ( a t l e a s t 10% o f t h e c e l l s l o c a t e d ) a t l o w s u r v i v a l l e v e l s ( l e s s t h a n 0 .5 ) when a c c u r a t e measurement o f s u r v i v a l 20 i s n o t as i m p o r t a n t . S u r v i v a l i s e s t i m a t e d a s f o l l o w s : S u r v i v a l , S = C E q u a t i o n 2 NP C=number o f s u r v i v o r s . N=C+D+E/2=number o f c e l l s p l a t e d . D=number o f d e a d c o l o n i e s . E=number o f p o s s i b l e e r r o r s . P=C/N a t z e r o r a d i a t i o n dose ( p l a t i n g e f f i c i e n c y ) . F o r e x p e r i m e n t s e t A , c e l l s were p l a t e d i n m i c r o t e s t p l a t e s . The a u t o m a t e d s y s t e m was u n f o r t u n a t e l y n o t a v a i l a b l e and c e l l s were l o c a t e d and c l a s s i f i e d m a n u a l l y t h r o u g h t h e m i c r o s c o p e u s i n g a l o c a t i o n c h a r t . F i g u r e 6 i s a t y p i c a l l o c a t i o n c h a r t u s e d b e f o r e t h e l o w d o s e a s s a y was a u t o m a t e d . 2 F o r e x p e r i m e n t s e t B c e l l s were p l a t e d i n 25 cm f l a s k s . Improvements t o t h e l o w d o s e a s s a y p r o c e d u r e u s e d f o r e x p e r i m e n t s e t B a r e d e s c r i b e d i n d e t a i l i n s e c t i o n 2 . 4 . F o r t h e i m p r o v e d p r o c e d u r e a c o m p u t e r i s u s e d t o c o n t r o l a m o t o r d r i v e n m i c r o s c o p e s t a g e t o a i d t h e e x p e r i m e n t e r i n t h e l o c a t i o n and t h e c l a s s i f i c a t i o n o f c e l l s . The l o w d o s e a s s a y i s d i s c u s s e d i n c o n s i d e r a b l y more d e t a i l by B r o s i n g i n h e r PhD t h e s i s , 1 9 8 3 . 2 . 4 THE AUTOMATED LOW DOSE ASSAY SYSTEM (ALDAS) T h i s s e c t i o n d e s c r i b e s t h e h a r d w a r e and s o f t w a r e d e v e l o p e d f o r ALDAS a t t h e B r i t i s h C o l u m b i a C a n c e r R e s e a r c h C e n t r e , V a n c o u v e r , B . C . (BCCRC) . I n s p i r a t i o n f o r t h e d e v e l o p m e n t o f t h i s s y s t e m came i n p a r t f r o m D r s . L . B r u b y and J . N e l s o n o f B a t t e l e , R i c h l a n d , W a s h i n g t o n , D r . R. K a l l m a n o f t h e S t a n f o r d U n i v e r s i t y S c h o o l o f M e d i c i n e , S t a n f o r d , C a l i f o r n i a , (H. Kemper e t a l , 1 9 8 0 ) , and D r . T o l m a c h o f W a s h i n g t o n U n i v e r s i t y S c h o o l o f M e d e c i n e , S t . L o u i s , M i s s o u r i , (R. Heye e t a l , 1 9 8 1 ) , who have d e v e l o p e d s i m i l a r s y s t e m s f o r t i m e l a p s e p h o t o g r a p h y . 21 Figure 6: LOW DOSE ASSAY LOCATION CHART. This a the chart used to record the locations and classifications of the cells assayed for survival with the low dose assay. Irradiated c e l l s were placed in microtest plates (figure 5) and located under the microscope 2-3 days following irradiation. Each dot (") on the chart corresponds to a located c e l l . C e l l locations were re-examined and c e l l classifications were recorded on the chart 6-8 days following irradiation. The symbols on the chart have the following meanings: C-'survivor', a colony with more than 50 c e l l s ; D-'killed', a colony with less than 50 c e l l s , • -'possible error', a c e l l cannot be found at the recorded location, and /^-'coincidence', colonies have overlapped. 22 2 . 4 . 1 ALDAS h a r d w a r e . F i g u r e 7 i s a p h o t o g r a p h o f t h i s s y s t e m . The b a s i c s y s t e m h a r d w a r e d e s c r i b e d b e l o w i s shown s c h e m a t i c a l l y i n f i g u r e 8 . The c o r e o f ALDAS i s a ZE ISS ICM 405 m i c r o s c o p e w i t h f a c i l i t y f o r m o u n t i n g a t e l e v i s i o n c a m e r a , 35 mm c a m e r a , and p o l a r o i d c a m e r a . A l t h o u g h s u c h a h i g h q u a l i t y m i c r o s c o p e i s n o t a r e q u i r e m e n t f o r ALDAS t h e a d v a n t a g e s t h a t one o b t a i n s f r o m image q u a l i t y , s t u r d i n e s s o f c o n s t r u c t i o n , and t h e image r e c o r d i n g f a c i l i t i e s a r e s i g n i f i c a n t . The m i c r o s c o p e s t a g e was t h e most d i f f i c u l t s y s t e m component t o b u i l d . The s t a g e must g l i d e s m o o t h l y t o m i n i m i z e f r i c t i o n so t h a t t h e s t e p p i n g m o t o r s w i l l n o t be o v e r l o a d e d , i n w h i c h c a s e t h e y may m i s s s t e p s . A n u t w h i c h a t t a c h e d t o e a c h l e a d s c r e w m u s t b e l i n k e d t o t h e s t a g e . T h i s n u t t r a n s l a t e s t h e r o t a t i o n a l m o t i o n o f t h e s c r e w s t o t h e t r a n s l a t i o n a l m o t i o n o f t h e s t a g e . The l e a d s c r e w s must be p r e c i s i o n mounted on t h e s t a g e so t h a t t h e r e i s a minimum o f f r i c t i o n d e v e l o p e d b e t w e e n t h e l e a d s c r e w and t h e n u t . I f t h e m o t o r s c a n n o t e a s i l y overcome t h i s f r i c t i o n t h e y w i l l m i s s s t e p s . The n u t must be f i r m l y a t t a c h e d t o t h e s t a g e s i n c e any ' l o o s e n e s s ' w i l l r e s u l t i n a l o s s o f r e p e a t a b i l i t y . The m i c r o s c o p e s t a g e was d e s i g n e d and b u i l t by Semprex c o r p o r a t i o n , 587 D i v i s i o n S t r e e t , C a m p b e l l , C a l i f o r n i a . I t was n e c e s s a r y t o have a m a c h i n i s t m o d i f y t h e s t a g e so t h a t i t c o u l d be a d a p t e d t o o u r m i c r o s c o p e . A m e r i c a n M o t i o n P r o d u c t 1.8° p e r s t e p s t e p p i n g m o t o r s a r e mounted on t h e s t a g e l e a d s c r e w s p r o v i d i n g a 10 um s t a g e movement p e r m o t o r s t e p w i t h l e s s t h a n 100 um o r 10 s t e p s b a c k l a s h . I n o r d e r t o a c h i e v e 10 um r e p e a t a b i l i t y i t w o u l d h a v e b e e n n e c e s s a r y t o a l w a y s a p p r o a c h t h e r e c o r d e d l o c a t i o n s f r o m t h e same d i r e c t i o n ; however f o r o u r p u r p o s e s 100 um r e p e a t a b i l i t y was s a t i s f a c t o r y . The b a c k l a s h was due p r i m a r i l y t o a l o o s e n e s s i n t h e l i n k b e t w e e n t h e l e a d s c r e w n u t and t h e 2 3 . Figure 7 : PHOTOGRAPHS OF ALDAS. These photographs show the hardware components of the automated low dose assay system. 24 Com.pu.i&r joystick. spco-kcr Q + Q 'iou$hcck a-neL J°<j •stick buJrtons :ors Microscope. s k o L j e , s i f A <> e micro swi •ic-hc s Figure 8: SCHEMATIC OF ALDAS. The following i s a t y p i c a l sequence of events: The computer sends an analog s i g n a l to the j o y s t i c k speaker through the D+7AI/0 board to s i g n a l the experimenter with an audible 'beep' that the microscope stage i s i n his/her c o n t r o l . The experimenter, wishing to move the stage, moves the j o y s t i c k which sends a d i g i t a l s i g n a l to the computer through the D+7AI/0 board which represents the magnitude of the speed to move the stage. The computer moves the stage by sending t r a n s i s t o r - t r a n s i s t o r l o g i c (TTL) pulses at the appropriate frequency to the microscope stage d r i v e r through the D+7AI/0 board. For each TTL pulse received the stage d r i v e r v a r i e s the current to the four windings of each motor i n the appropriate sequence to rotate the motor one step, moving the stage 10 um. The stage microswitches prevent stage overrun by s i g n a l l i n g the computer when they are depressed. 25 s t a g e . The b a c k l a s h o f t h e s t a g e h a s s i n c e b e e n i m p r o v e d t o a b o u t two m o t o r s t e p s by r e p l a c i n g t h e two p l a s t i c n u t s w h i c h l i n k e d t h e l e a d s c r e w s t o t h e s t a g e w i t h two c a r e f u l l y m a c h i n e d b r a s s n u t s . The s t e p p i n g m o t o r s a r e e a c h d r i v e n by a SLOSYN STM-103 t r a n s l a t o r modu le w h i c h p r o v i d e s c u r r e n t f r o m t h e Hammond 24 v o l t , 2 . 2 ampere power s u p p l i e s t o e a c h o f t h e f o u r m o t o r w i n d i n g s i n t h e c o r r e c t s e q u e n c e t o move t h e m o t o r one s t e p i n e i t h e r t h e c l o c k w i s e (CW) o r c o u n t e r c l o c k w i s e (CCW) d i r e c t i o n . The t r a n s l a t o r modu le h a s i t s own i n t e r n a l o s c i l l a t o r so t h a t t h e m o t o r s may be d r i v e n d i r e c t l y w i t h t h e t r a n s l a t o r m o d u l e s o r w i t h an i n v e r t e d t r a n s i s t o r - t r a n s i s t o r l o g i c (TTL) p u l s e f r o m a m i c r o p r o c e s s o r . The o p e r a t i o n o f t h e t r a n s l a t o r m o d u l e s i s d e s c r i b e d i n t h e S u p e r i o r E l e c t r i c Company m a n u a l , " I n s t r u c t i o n s f o r I n s t a l l a t i o n , o p e r a t i o n , and M a i n t e n a n c e o f SLO -SYN T r a n s l a t o r Modu le Type STM 1 0 3 " . We have d e s i g n e d a b o x w h i c h h o u s e s t h e t r a n s l a t o r m o d u l e s , power s u p p l i e s , and some a d d i t i o n a l l o g i c u s e d t o p r e v e n t s i m u l t a n e o u s i n p u t o f p u l s e s f r o m t h e c o m p u t e r and e i t h e r o f t h e t r a n s l a t o r modu le o s c i l l a t o r s and t o p r e v e n t t h e m o t o r s f r o m s t e p p i n g when t h e l i m i t s w i t c h e s mounted on t h e m i c r o s c o p e s t a g e have b e e n r e a c h e d . F i g u r e 9 i s a s c h e m a t i c o f t h i s i n t e r f a c e . I w o u l d l i k e t o a c k n o w l e d g e t h e h e l p so f r e e l y g i v e n b y I v a n L i u and D r . G a b r i e l Lam o f t h e b i o m e d i c a l g r o u p o f TRIUMF, U n i v e r s i t y o f B r i t i s h C o l u m b i a , i n t h e d e s i g n i n g and w i r i n g o f t h e c i r c u i t r y f o r t h i s b o x . The t r a n s l a t o r m o d u l e s a r e i n t e r f a c e d t o a CROMEMCO SYSTEM-THREE c o m p u t e r t h r o u g h a CROMEMCO D+7AI / 0 b o a r d . T h i s b o a r d p r o v i d e s s e v e n c h a n n e l s o f d i g i t a l - t o - a n a l o g c o n v e r s i o n , s e v e n c h a n n e l s o f a n a l o g - t o - d i g i t a l c o n v e r s i o n and one p a r a l l e l i n p u t - o u t p u t p o r t . F o u r b i t s o f t h e p a r a l l e l I /O p o r t a r e i n t e r f a c e d t o t h e two t r a n s l a t o r m o d u l e s p r o v i d i n g CW and CCW m o t i o n f o r b o t h m o t o r s . 26 F i g u r e 9 : SCHEMATIC OF THE MICROSCOPE STAGE DRIVER. The d r i v e r c a n be u s e d t o r u n t h e s t a g e m o t o r s w i t h o r w i t h o u t a c o m p u t e r . The c o m p u t e r i s i n t e r f a c e d t o t h e d r i v e r t h r o u g h t h e D+7AI /0 b o a r d . The d i g i t a l s i g n a l f r o m t h e D+7AI /0 b o a r d d e f i n e s w h i c h m o t o r i s t o move and i n w h i c h d i r e c t i o n , c o u n t e r c l o c k w i s e (CCW) o r c l o c k w i s e (CW). The STM-103 t r a n s l a t o r m o d u l e s c o n v e r t e a c h TTL p u l s e s e n t f r o m t h e b o a r d i n t o a s e q u e n c e o f p u l s e s w h i c h p r o v i d e c u r r e n t t o e a c h o f t h e f o u r m o t o r w i n d i n g s i n t h e c o r r e c t s e q u e n c e t o move t h e m o t o r one s t e p . The m i c r o s w i t c h e s p r e v e n t s t a g e o v e r r u n . 27 A CROMEMCO J S - 1 j o y s t i c k i s i n t e r f a c e d t o t h e c o m p u t e r t h r o u g h t h e D+7AI /0 b o a r d . The j o y s t i c k c o n s o l e h o u s e s a j o y s t i c k , f o u r b u t t o n s , and a s p e a k e r . The j o y s t i c k i s u s e d t o c o n t r o l t h e s p e e d and d i r e c t i o n o f t h e m i c r o s c o p e s t a g e . The f o u r b u t t o n s a r e u s e d f o r i n p u t o f a l t e r n a t i v e s t o p r o v i d e b r a n c h i n g i n f o r m a t i o n t o t h e s o f t w a r e . T h e s e b u t t o n s a r e a l s o u s e d f o r t h e i n p u t o f l o c a t i o n c l a s s i f i c a t i o n s . The s p e a k e r i s u s e d t o s i g n a l t h e o b s e r v e r f o r i n p u t f r o m t h e j o y s t i c k c o n s o l e . The j o y s t i c k i s p l u g g e d i n t o t h e b o x w h i c h h o u s e s t h e t r a n s l a t o r m o d u l e s . I n t h i s way o n l y one c a b l e i s r e q u i r e d t o c o n n e c t ALDAS t o t h e compute r and ALDAS c a n be o p e r a t e d r e m o t e f r o m t h e c o m p u t e r , f o r examp le i n a l a m i n a r f l o w hood o r i n a warm room. The m a n u f a c t u r e r , s p e c i f i c a t i o n s , and c o s t o f t h e s y s t e m components a r e s u m m a r i z e d i n t a b l e 1 . 2 . 4 . 2 ALDAS s o f t w a r e . The j o y s t i c k , m e c h a n i c a l s t a g e m i c r o s w i t c h e s , and t h e s t a g e m o t o r s o f ALDAS a r e i n t e r f a c e d t h r o u g h t h e m i c r o s c o p e s t a g e d r i v e r t o t h e CROMEMCO D+7AI /0 b o a r d o f t h e CROMEMCO c o m p u t e r . The s o f t w a r e c o m m u n i c a t e s w i t h t h e s e p e r i p h e r a l d e v i c e s by s e n d i n g o r r e c e i v i n g b y t e s t o t h e a p p r o p r i a t e p o r t a d d r e s s on t h e D+7AI /0 b o a r d . The b o a r d c o n s i s t s o f s e v e n c h a n n e l s o f 8 - b i t A / D c o n v e r s i o n and s e v e n c h a n n e l s o f D /A c o n v e r s i o n and one s e v e n - b i t p a r a l l e l I / O . These a n a l o g p o r t s and t h e d i g i t a l p o r t a r e a c c e s s e d as i n p u t and o u t p u t p o r t s o f t h e c o m p u t e r s y s t e m . F i g u r e 10 i s a l i s t o f t h e c o m p u t e r s y s t e m p o r t s o f t h e D+7AI /0 b o a r d u t i l i z e d by ALDAS. The j o y s t i c k , b u t t o n s , and s p e a k e r on t h e j o y s t i c k box a l l commun ica te d i r e c t l y w i t h t h e D+7AI /0 b o a r d . The d i g i t a l s i g n a l f r o m t h e b u t t o n s i s i n p u t t h r o u g h t h e p a r a l l e l p o r t , t h e a n a l o g s i g n a l s f r o m t h e X and Y a x i s o f t h e j o y s t i c k a r e i n p u t t h r o u g h a n a l o g p o r t one and two r e s p e c t i v e l y , and t h e a n a l o g s i g n a l t o t h e s p e a k e r i s o u t p u t t h r o u g h a n a l o g p o r t one o f t h e D+7AI /0 b o a r d . The X a x i s o f t h e j o y s t i c k i s d e f i n e d 28 TABLE I TABLE I : L i s t o f t h e components o f ALDAS. The h a r d w a r e components o f ALDAS a r e l i s t e d a l o n g w i t h t h e i r m a n u f a c t u r e r , s p e c i f i c a t i o n s , and a p p r o x i m a t e c o s t i n C a n a d i a n d o l l a r s a t t h e t i m e o f p u r c h a s e , 1982 . COMPONENT MANUFACTURER SPECIFICATIONS COST (Canad ian ) m i c r o s c o p e ZEISS M o d e l ICM-405 m e c h a n i c a l s t a g e SEMPREX 2 m m / r e v . o f s c r e w , 7 cm x 12 cm t r a v e l . $3000 s t e p p i n g m o t o r s A p p l i e d M o t i o n 12V, 0 . 1 6 Amp p e r w i n d i n g , $300 P r o d u c t s 1.8° p e r s t e p power s u p p l i e s HAMMOND 2 . 4 V o l t , 2 . 2 Amp. $92 e a . m o t o r t r a n s l a t o r s SLO SYN Model STM-103. Up to 3000 steps/sec. Has i n t e r n a l o s c i l l a t o r . Accept TTL pulse f o r motor step. $240 e a . ALDAS i n t e r f a c e CROMEMCO M o d e l D+7AI /0 b o a r d . 7 D /A and 7 A / D p o r t s . One p a r a l l e l I / O p o r t . $475 j o y s t i c k CROMEMCO 2-D v a r i a b l e p o t . 0 - 5 V o l t s , 4 s w i t c h e s , 0 - 5 V . S p e a k e r . $235 c o m p u t e r CROMEMCO 48 K w o r k i n g s p a c e . (o r e q u i v a l e n t ) 2 - 8 " f l o p p y d i s k s , Z - 8 0 CPU. 4 MHz o p e r a t i o n . $3000 29 D+7AI/0 port P a r a l l e l port. Assignment b i t s 0-3 are the output of buttons 1-4 on the jo y s t i c k console. b i t s 4-7 are the output of the microswitches which prevent stage overrun. b i t 0 i s the input to the X motor, clockwise. b i t 1 i s the input to the X motor, counter-clockwise. Comment b i t b-l=0 when button b i s depressed. Otherwise b i t b - l = l . bit=0 when corresponding microswitch i s depressed i n d i c a t i n g stage overrun. one inverted TTL pulse per motor step. one inverted TTL pulse per motor step. b i t 2 i s the input to the Y motor, clockwise. b i t 3 i s the input to the Y motor, counter-clockwise. one inverted TTL pulse per motor step. one inverted TTL putse per motor step. Analog port 1 Analog port 2 input from the X axis of the j o y s t i c k . output to the j o y s t i c k speaker, t input from the Y axis of the j o y s t i c k . I/O i s an integer from -128 to 127. I/O i s an integer from -128 to 127. I/O i s an integer from -128 to 127. Figure 10: I/O PORTS ON THE ANALOG TO DIGITAL CONVERTER. The CROMEMCO D+7AI/0 board i s int e r f a c e d to the CROMEMCO computer (figure 8) . The board has one p a r a l l e l I/O (input/output) port and seven A/D (analog to d i g i t a l ) and seven D/A converters. Only the d i g i t a l port, two of the A/D ports and one of the D/A ports i s u t i l i z e d by ALDAS. 30 t o be i n c r e a s i n g p o s i t i v e f r o m l e f t t o r i g h t and t h e Y a x i s i n c r e a s i n g p o s i t i v e f r o m f r o n t t o b a c k . The s t e p p i n g m o t o r s communicate w i t h t h e D+7AI /0 b o a r d t h r o u g h t h e m i c r o s c o p e s t a g e d r i v e r . The m i c r o s c o p e s t a g e d r i v e r c o n t a i n s t h e power s u p p l y f o r e a c h m o t o r and t h e l o g i c n e c e s s a r y t o c o n v e r t a TTL p u l s e i n t o t h e c o r r e c t sequence o f p u l s e s t o t h e m o t o r w i n d i n g s . The m o t o r w i n d i n g s draw 160 mA f r o m t h e power s u p p l y when s w i t c h e d o n . T h e r e a r e f o u r i n p u t p o r t s on t h e m i c r o s c o p e s t a g e d r i v e r f o r m o v i n g t h e X m o t o r c l o c k w i s e (CW), t h e X m o t o r c o u n t e r c l o c k w i s e (CCW), t h e Y m o t o r c l o c k w i s e (CW), and t h e Y m o t o r c o u n t e r c l o c k w i s e (CCW). These a r e i n t e r f a c e d t o b i t s 0 - 3 r e s p e c t i v e l y o f t h e D+7AI /0 p a r a l l e l p o r t . The d r i v e r i s t r i g g e r e d by a change f r o m l o g i c l e v e l 0 (0 t o 0 . 4 V o l t s DC) t o l o g i c l e v e l 1 ( 2 . 0 t o 5 . 2 5 V o l t s D C ) , t h e t r a i l i n g edge o f a TTL p u l s e . LDA i s a s e t o f i n t e r a c t i v e F o r t r a n p r o g r a m s d e v e l o p e d f o r ALDAS. Of t h e t h r e e p r o g r a m m i n g l a n g u a g e s a v a i l a b l e , B A S I C , Z - 8 0 a s s e m b l e r , and F o r t r a n , t h e l a t t e r o f f e r e d t h e g r e a t e s t v e r s a t i l i t y and e a s e i n p r o g r a m m i n g and I / O and t h e c o m p i l e d v e r s i o n was s u f f i c i e n t l y f a s t t o w a r r a n t i t s u s e . A c o m p r e h e n s i v e u s e r ' s g u i d e h a s b e e n w r i t t e n f o r t h e s e p r o g r a m s . ( Faddegon , 1 9 8 2 ) . The LDA p r o g r a m s u t i l i z e two m a j o r s u b r o u t i n e s . The m a n u a l s t a g e movement s u b r o u t i n e , MSCAN, p r o v i d e s f o r c o n t r o l o f t h e d i r e c t i o n and s p e e d o f t h e m i c r o s c o p e s t a g e t h r o u g h t h e j o y s t i c k . The v a l u e o f t h e j o y s t i c k i n p u t d e f i n e s t h e number o f t i m e s (0 -64) e a c h o f b i t 0 - 3 o f t h e c o m p u t e r s y s t e m I / O p o r t 24 i s s e t t o z e r o r e s u l t i n g i n a m o t o r s t e p b e f o r e t h e j o y s t i c k i s r e a d a g a i n . The j o y s t i c k b u t t o n s a r e u s e d t o r e t u r n f r o m t h e s u b r o u t i n e and p r o v i d e b r a n c h i n g i n f o r m a t i o n t o t h e m a i n r o u t i n e . 31 The a u t o m a t i c s t a g e movement s u b r o u t i n e , ASCAN, p r o v i d e s f o r c o n t r o l o f t h e s p e e d o f t h e m i c r o s c o p e s t a g e t h r o u g h t h e j o y s t i c k . The v a l u e o f t h e j o y s t i c k i n p u t d e f i n e s t h e l e n g t h o f t i m e t h a t a c l o c k r u n s b e f o r e t h e n e x t p u l s e i s o u t p u t . D i r e c t i o n a l and d i s t a n c e c o n t r o l i s p r o v i d e d t h r o u g h t h e s o f t w a r e . Once t h e s t a g e h a s r e a c h e d i t s d e s t i n a t i o n , t h e p r o g r a m r e t u r n s f r o m t h e s u b r o u t i n e . The j o y s t i c k b u t t o n s may be u s e d t o r e t u r n f r o m t h e s u b r o u t i n e and p r o v i d e b r a n c h i n g i n f o r m a t i o n t o t h e m a i n r o u t i n e . The FORTRAN c o d i n g o f MSCAN and ASCAN i s l i s t e d i n t h e a p p e n d i x a l o n g w i t h t h e v a r i a b l e s u s e d i n t h e s u b r o u t i n e s . T h r e e o t h e r s u b r o u t i n e s were r e q u i r e d . S u b r o u t i n e BEEP was u s e d t o s i g n a l t h e o p e r a t o r t h r o u g h t h e j o y s t i c k s p e a k e r . S u b r o u t i n e s PL INE and OUT50 were u s e d t o p r i n t f i l e s i n t e r a c t i v e l y f r o m t h e F o r t r a n p r o g r a m s LDALOC and LDACLASS. 2 . 4 . 3 The c e l l l o c a t i n g p r o g r a m LDALOC. LDALOC i s u s e d t o l o c a t e c e l l s . F i g u r e 11 i s a s i m p l i f i e d f l o w c h a r t o f p r o g r a m LDALOC. On e n t e r i n g t h e p r o g r a m t h e u s e r i s p r o v i d e d w i t h t h e f o l l o w i n g s e t o f o p t i o n s : LOCATION SCAN MENU: A : S t a r t r e g u l a r s c a n . (User c o n t r o l s s c a n s p e e d . S c a n p o i n t s a r e u s e r s u p p l i e d o r c o m p u t e r g e n e r a t e d ) . B : S t a r t d i s c r e t e p o i n t s c a n . (S tage moved a t maximum s p e e d b e t w e e n c o m p u t e r g e n e r a t e d s c a n p o i n t s ) . C : D e f i n e s c a n b o u n d a r i e s , r e f e r e n c e p o i n t , and s c a n p o i n t s i f a n y . D: R e d e f i n e s c a n p a r a m e t e r s . E : S u b m i t p r i n t o u t t o p r i n t e r . Z : E x i t r o u t i n e . The l o c a t i o n s c a n menu o p t i o n s a r e e n t e r e d f r o m t h e c o m p u t e r c o n s o l e . O p t i o n Z r e t u r n s t h e u s e r t o CDOS, t h e CROMEMCO d i s k o p e r a t i n g s y s t e m . 32 Sea no-L "to cam.pu.icr ~6o record. 6t>& current siaje (celt)Location. Centre, cell In fieLoi of vietti. IJKO-C "6o coMptcter to record-u-rre/it st&je Loca~-trc.cn.. ^Centre celL Ln field of vieoJ. Use. the jot/slick "ho control, 'ike. speed, of the sta-cje. dohiue It moves tothe. n&Yjb SCa.fi poln~t' .orx,pu.ie.r oudo'X'O-ilcailtj /naves •the staje. o,~t top speed. 60 -the ficx.t scan, point.. FINISH Locate dlauoKallu apposite, boa/tdaru corners o/t, -£he celt flask -tt-nol -£Jie -flask reference position. a-sitj ifie joys£ick. Compu-ler lists the Scan paro-irre.terSj available ranges, O-itd curren-t values. Jznler -ike neu> value, of tne paro.rn.eter. Ii He contpaier generate iXe scon.poCn.xs\ 'NO (dse the. Joystick £oca.ie. ts. Figure 11: LDALOC FLOWCHART. LDALOC i s the location scan program for the automated low dose assay system ALDAS. This figure i s a 'bare bones' flowchart of the possible uses of this program from a user's point of view. Ovals represent branch points and squares show the procedure followed by the experimenter or the computer does the program branches. For example, ce l l s can be located and their locations recorded by branching to either option A or option B. 33 O p t i o n A : O p t i o n s A and B a r e u s e d t o l o c a t e c e l l s i n t h e v e s s e l and t o r e c o r d t h e s e l o c a t i o n s . The u s e r p o s i t i o n s t h e s t a g e a t t h e r e f e r e n c e p o s i t i o n d e f i n e d i n o p t i o n C. The p r o g r a m t h e n c a u s e s t h e s t a g e t o move b e t w e e n t h e s c a n p o i n t s . These c a n be s u p p l i e d by t h e u s e r t h r o u g h o p t i o n C o r c a n be c o m p u t e r g e n e r a t e d . R e f e r t o o p t i o n C f o r d e t a i l s . As t h e s t a g e moves t o t h e n e x t s c a n p o i n t t h e u s e r c o n t r o l s t h e s p e e d o f t h e s c a n a n d i s p r o v i d e d w i t h t h e f o l l o w i n g a l t e r n a t i v e s t o b e e n t e r e d f r o m t h e j o y s t i c k b u t t o n s : A u t o m a t i c S c a n A l t e r n a t i v e s : 1 ,2 - Go t o m a n u a l s c a n 3 - D e l e t e l a s t r e c o r d e d l o c a t i o n 4 - R e t u r n s t a g e t o r e f e r e n c e p o s i t i o n and e x i t . A l t e r n a t i v e 3 d e c r e m e n t s t h e r e c o r d e d l o c a t i o n c o u n t e r NLOC, w h i c h i s e q u i v a l e n t t o d e l e t i n g t h e l a s t r e c o r d e d l o c a t i o n a l o n g w i t h i t s c l a s s i f i c a t i o n . T h i s a l t e r n a t i v e i s u s e f u l i f an e r r o r was made i n t h e l o c a t i o n o r c l a s s i f i c a t i o n o f a c e l l . A l t e r n a t i v e 4 o u t p u t s t h e r e c o r d e d l o c a t i o n s s t o r e d i n c o m p u t e r memory t o t h e f l o p p y d i s k t o t h e f i l e s " F i l e n a m e . L D A " , where F i l e n a m e i s u s e r s u p p l i e d , and "LDAPRINT.OUT" . The c o m p u t e r t h e n r e t u r n s t h e m i c r o s c o p e s t a g e t o t h e r e f e r e n c e p o s i t i o n and p r i n t s o u t t h e - L o c a t i o n S c a n Menu. A l t e r n a t i v e s 1 and 2 s t o p t h e s c a n b e t w e e n t h e s c a n p o i n t s . The u s e r c a n r e p o s i t i o n t h e s t a g e w i t h t h e j o y s t i c k and e n t e r one o f t h e f o l l o w i n g a l t e r n a t i v e s f r o m t h e j o y s t i c k b u t t o n s : M a n u a l S c a n A l t e r n a t i v e s : 1 - R e c o r d l o c a t i o n . 2 - R e c o r d l o c a t i o n w i t h c l a s s i f i c a t i o n . 3 - C o i n c i d e n c e c h e c k . 4 - Resume s c a n . 34 A l t e r n a t i v e s 1 and 2 i n c r e m e n t t h e l o c a t i o n c o u n t e r , NLOC, and r e c o r d t h e c u r r e n t s t a g e l o c a t i o n i n c o m p u t e r memory. A l t e r n a t i v e 2 a l l o w s t h e u s e r t o r e c o r d a c l a s s i f i c a t i o n o f t h e c e l l s f o u n d a t t h e r e c o r d e d l o c a t i o n . A l t e r n a t i v e 3 c h e c k s t o see i f any p r e v i o u s l y r e c o r d e d l o c a t i o n s a r e w i t h i n a u s e r d e f i n e d r e g i o n o f t h e c u r r e n t s t a g e l o c a t i o n . (See o p t i o n D o f t h e L o c a t i o n S c a n Menu. ) I f s o , t h e s t a g e i s moved t o t h o s e l o c a t i o n s . I n any c a s e , no l o c a t i o n i s r e c o r d e d and t h e u s e r i s a g a i n p r o v i d e d w i t h t h e m a n u a l s c a n a l t e r n a t i v e s . A l t e r n a t i v e 4 c a u s e s a r e s u m p t i o n o f t h e s c a n t o t h e n e x t s c a n p o i n t . The u s e r i s p r o v i d e d w i t h t h e a u t o m a t i c s c a n a l t e r n a t i v e s . O p t i o n B : The s c a n p o i n t s f o r o p t i o n B a r e c o m p u t e r g e n e r a t e d . The s t a g e i s moved a t maximum s p e e d t o w a r d s e a c h s c a n p o i n t i n t u r n . The s t a g e i s s t o p p e d e v e r y t i m e i t h a s moved a f i x e d i n t e r v a l as d e f i n e d by t h e u s e r t h r o u g h o p t i o n D. The u s e r i s t h e n p r o v i d e d w i t h t h e f o l l o w i n g s e t o f a l t e r n a t i v e s : D i s c r e t e P o i n t S c a n A l t e r n a t i v e s : 1 - R e c o r d l o c a t i o n . 2 - Go t o manua l s c a n . 3 - Resume s c a n . 4 - R e t u r n s t a g e t o r e f e r e n c e p o s i t i o n and e x i t . A l t e r n a t i v e 1 r e c o r d s t h e c u r r e n t s t a g e l o c a t i o n i n c o m p u t e r memory. The s t a g e i s t h e n moved t o t h e n e x t l o c a t i o n a t maximum s p e e d and t h e u s e r i s p r o v i d e d w i t h t h e d i s c r e t e p o i n t s c a n a l t e r n a t i v e s . A l t e r n a t i v e 2 a l l o w s t h e u s e r t o r e p o s i t i o n t h e s t a g e w i t h t h e j o y s t i c k and e n t e r one o f t h e m a n u a l s c a n a l t e r n a t i v e s . These a r e t h e same as f o r o p t i o n A . A l t e r n a t i v e 3 moves t h e s t a g e t o t h e n e x t l o c a t i o n ( w i t h o u t r e c o r d i n g t h e c u r r e n t l o c a t i o n ) and p r o v i d e s t h e u s e r w i t h t h e d i s c r e t e p o i n t s c a n a l t e r n a t i v e s . A l t e r n a t i v e 4 o u t p u t s t h e r e c o r d e d l o c a t i o n s s t o r e d i n c o m p u t e r memory t o 35 f i l e s " F i l e n a m e . L D A " and "LDAPRINT.OUT" on t h e f l o p p y d i s k . O p t i o n C : O p t i o n C i s u s e d t o d e f i n e t h e f o l l o w i n g p a r a m e t e r s : 1 . The r e c t a n g u l a r b o u n d a r y b e y o n d w h i c h t h e s t a g e i s n o t p e r m i t t e d t o move by t h e s o f t w a r e . 2 . The r e f e r e n c e p o s i t i o n f r o m w h i c h t h e l o c a t i o n and c l a s s i f i c a t i o n s c a n s w i l l b e g i n . 3 . The s c a n p o i n t s ( i f any) f o r t h e l o c a t i o n s c a n . I f no s c a n p o i n t s a r e i n p u t a g r i d o f s c a n p o i n t s w i l l be p r o v i d e d by t h e s o f t w a r e . O p t i o n C n e e d o n l y b e u s e d o n c e f o r e a c h t y p e o f v e s s e l ( m i c r o t e s t p l a t e , f l a s k , p e t r i ) s c a n n e d . The d a t a i s o u t p u t t o a f i l e "LDALOCn.DAT" on t h e A d i s k , where n i s a u s e r s u p p l i e d p r o g r a m p a r a m e t e r d e f i n e d u s i n g o p t i o n D. T h i s f i l e i s r e w r i t t e n e v e r y t i m e t h e s c a n p o i n t s a r e o u t p u t f r o m o p t i o n C . The u s e r may have up t o t e n s u c h f i l e s (n = 0, 9) f o r d i f f e r e n t v e s s e l s o r d i f f e r e n t s c a n p a r a m e t e r s . O p t i o n D: O p t i o n D i s u s e d t o l i s t a n d / o r r e d e f i n e t h e u s e r d e f i n a b l e p r o g r a m p a r a m e t e r s . These a r e l i s t e d b e l o w : PARAMETER RANGE COMMENT A : SPEED 0 - 1 0 M o t o r s p e e d , 10 = maximum B : CELL COUNT 10 - 400 Max. n o . o f l o c a t i o n s t o be r e c o r d e d . C : COINCIDENCE CHECK 0 - 1 0 = A u t o m a t i c c o i n c i d e n c e c h e c k 1 = C o i n c i d e n c e c h e c k f r o m MANUAL SCAN ALTERNATIVES D: COINCIDENCE RANGE 0 - 127 L e n g t h o f s q u a r e r e g i o n i n w h i c h ' t o s e a r c h f o r c o i n c i d e n c e s . SCAN PATTERN PARAMETERS E : L I N E SPACING 10 - 1270 Number o f s t e p s b e t w e e n s c a n l i n e s . F : POINT SPACING 10 - 1270 Number o f s t e p s b e t w e e n s c a n p o i n t s , d i s c r e t e p o i n t s c a n . A l s o d i s t a n c e b e t w e e n Y s c a n b o u n d a r i e s and t h e compute r g e n e r a t e d s c a n p o i n t s . G: DATA F I L E 0 - 9 Number, n , d e f i n i n g d a t a f i l e f o r p o i n t r e t r i e v a l , "LDALOCn.DAT" . P a r a m e t e r A c o n t r o l s t h e maximum s p e e d a t w h i c h t h e m o t o r s w i l l t u r n i n b o t h a u t o m a t i c (ASCAN) and manua l (MSCAN) s c a n modes. Too h i g h a v a l u e c a n 3 6 r e s u l t i n m i s s e d s t e p s , p a r t i c u l a r l y a t t h e edges o f t h e s c a n o r i f t h e s t a g e i s m o v i n g a h e a v y l o a d . A v a l u e o f 8 i s recommended f o r n o r m a l u s e . P a r a m e t e r B l i m i t s t h e number o f l o c a t i o n s t o be r e c o r d e d d u r i n g t h e s c a n u s i n g o p t i o n s A o r B . I f p a r a m e t e r B h a s a v a l u e o f 100 t h e l o c a t i o n s c a n w i l l be t e r m i n a t e d a f t e r 100 l o c a t i o n s have b e e n r e c o r d e d . P a r a m e t e r D d e f i n e s t h e r e g i o n i n w h i c h p r e v i o u s l y r e c o r d e d l o c a t i o n s w i l l be c o n s i d e r e d c o i n c i d e n t w i t h t h e c u r r e n t l o c a t i o n . I f p a r a m e t e r D h a s a v a l u e o f 5 0 , t h e c o i n c i d e n c e r e g i o n i s 100 s t e p s l o n g by 100 s t e p s w i d e c e n t e r e d a t t h e c u r r e n t l o c a t i o n . P a r a m e t e r E d e f i n e s t h e number o f s t e p s b e t w e e n s c a n l i n e s when t h e s c a n p o i n t s a r e s o f t w a r e g e n e r a t e d . P a r a m e t e r F d e f i n e s t h e d i s t a n c e b e t w e e n t h e Y s c a n b o u n d a r i e s and t h e n e a r e s t s c a n p o i n t s when t h e s c a n p o i n t s a r e s o f t w a r e g e n e r a t e d . P a r a m e t e r F a l s o d e f i n e s t h e i n t e r v a l t o move t h e s t a g e d u r i n g t h e d i s c r e t e p o i n t s c a n ( o p t i o n B ) . P a r a m e t e r G d e f i n e s t h e s e v e n t h c h a r a c t e r , n , o f t h e f i l e n a m e o f t h e d a t a f i l e , "LDALOCn.DAT" , w h i c h i s u s e d t o s t o r e t h e r e f e r e n c e i n f o r m a t i o n f r o m o p t i o n C . O p t i o n E : O p t i o n E i s u s e d t o s u b m i t t h e d a t a i n f i l e "LDAPRINT.OUT" t o t h e l i n e p r i n t e r . T h i s f i l e c o n t a i n s a l i s t i n g o f t h e r e c o r d e d l o c a t i o n s a l o n g w i t h t h e i r c l a s s i f i c a t i o n s f o r a l l o f t h e l o c a t i o n s c a n s p e r f o r m e d d u r i n g t h e c o m p u t e r s e s s i o n a f t e r t h e l a s t t i m e o p t i o n E was u s e d . A f t e r u s i n g o p t i o n E t h e c o n t e n t s o f "LDAPRINT.OUT" w i l l be e r a s e d and r e p l a c e d by t h e r e c o r d e d l o c a t i o n s f r o m s u b s e q u e n t l o c a t i o n s c a n s . 2 . 4 . 4 The c e l l c l a s s i f i c a t i o n p r o g r a m , LDACLASS. LDACLASS i s u s e d t o c l a s s i f y c e l l s . F i g u r e 12 i s a s i m p l i f i e d f l o w c h a r t o f LDACLASS. The u s e r i s p r o v i d e d w i t h t h e f o l l o w i n g s e t o f o p t i o n s : 37 CoMf>oL~te.r records 6fie. c C a t i i f i c a . 6 c o n of 6/ie, CoCotly frovidcd. &tj eVy pe.ri.jrt.e.n.'te.r. c prvpu-icr /r/avCS "ttit. s i a j e , to the. next ccuL 6oca.-6c.on.. START Compu.ie.r re.-6re.fvti iAt. cCassc/tco-icon olojia. Jrom. ~/C• <y>/?'ro/erca.i{re. &6t and. o-p 6cmel 6Ao\-6 &o\ck. c~C.txtsi-Kca,£i.on. occurs. FINISH Compa.ie.r prenyl oa.-6 £/e. tverU conf 6c.-6eol c/t £Acs SCSSCen.. Com. f>a- 6cr L^iS 6& £/& avACCa-ble. r-tx/iye-Sj a./>ol cttr-re.n.6 \s<z-6ues. Frxtzr Ike. p<xro-m t--6c r • Figure 1 2 : LDACLASS FLOWCHART. LDACLASS i s the clas s i f i c a t i o n scan program for the automated low dose assay system, ALDAS. This figure i s a 'bare bones' flowchart of the various uses of this program from a user's point of view. Ovals represent branch points and squares show the procedure followed by the computer or the experimenter after the program branches. For example, cells can be cla s s i f i e d and their classifications recorded by branching to option A. 38 CLASSIF ICATION SCAN MENU: A : S t a r t s c a n . C : C a l c u l a t e f r e q u e n c y o f c l a s s i f i c a t i o n . D: R e d e f i n e s c a n p a r a m e t e r s . E : S u b m i t p r i n t o u t t o p r i n t e r . Z : E x i t r o u t i n e . The c l a s s i f i c a t i o n s c a n menu o p t i o n s a r e e n t e r e d f r o m t h e c o m p u t e r c o n s o l e . O p t i o n Z r e t u r n s t h e u s e r t o CDOS, t h e Cromemco d i s k o p e r a t i n g s y s t e m . O p t i o n A : O p t i o n A i s u s e d t o c l a s s i f y c e l l s l o c a t e d w i t h LDALOC. The u s e r p o s i t i o n s t h e s t a g e a t t h e r e f e r e n c e p o s i t i o n f o r t h e v e s s e l . The p r o g r a m t h e n c a u s e s t h e s t a g e t o move i n t u r n t o e a c h l o c a t i o n r e t r i e v e d f r o m t h e f l o p p y d i s k f i l e " F i l e n a m e . L D A " where " F i l e n a m e " i s u s e r s u p p l i e d . The c o m p u t e r c a n be c o n s t r a i n e d t o move t h e s t a g e o n l y t o t h o s e l o c a t i o n s w i t h a g i v e n c l a s s i f i c a t i o n . (See o p t i o n D.) A f t e r e a c h l o c a t i o n i s r e a c h e d t h e u s e r i s p r o v i d e d w i t h t h e f o l l o w i n g s e t o f a l t e r n a t i v e s t o be e n t e r e d f r o m t h e j o y s t i c k b u t t o n s : C l a s s i f i c a t i o n S c a n A l t e r n a t i v e s : 1 - C l a s s i f y . 2 - Move s t a g e . 3 - Resume s c a n . 4 - E x i t r o u t i n e . I f a l t e r n a t i v e 1 i s s e l e c t e d t h e u s e r h a s an o p p o r t u n i t y t o c l a s s i f y t h e c o l o n y a t e a c h l o c a t i o n . A c l a s s i f i c a t i o n o f 1 -4 i s e n t e r e d u s i n g t h e j o y s t i c k b u t t o n s . I f a l o c a t i o n i s r e a c h e d i n LDACLASS b u t n o t c l a s s i f i e d b y t h e u s e r , ( c l a s s i f i c a t i o n s c a n a l t e r n a t i v e 3) i t i s g i v e n a c l a s s i f i c a t i o n o f 5 . The c o m p u t e r m a i n t a i n s a f i l e o f c e l l l o c a t i o n s and t h e i r c l a s s i f i c a t i o n s w h i c h a r e o u t p u t t o " F i l e n a m e . L D A " when t h e c l a s s i f i c a t i o n i s c o m p l e t e . Up t o f i v e s e t s o f c l a s s i f i c a t i o n s ( i n c l u d i n g 39 t h e c l a s s i f i c a t i o n i n LDALOC, w h i c h i s c l a s s i f i c a t i o n s e t one) a r e a l l o w e d p e r c e l l l o c a t i o n . The s i x c l a s s i f i c a t i o n s a r e d e f i n e d as f o l l o w s : C l a s s i f i c a t i o n D e f i n i t i o n 0 L o c a t i o n n e v e r r e a c h e d d u r i n g c l a s s i f i -c a t i o n s c a n . 1 - 4 U s e r d e f i n e d c l a s s i f i c a t i o n s f r o m LDALOC o r LDACLASS. 5 L o c a t i o n r e a c h e d d u r i n g c l a s s i f i c a t i o n s c a n b u t n o t c l a s s i f i e d by u s e r . The u s e r may r e c l a s s i f y any c l a s s i f i c a t i o n w i t h i n any c l a s s i f i c a t i o n s e t ( d e f a u l t i s f i r s t c l a s s i f i c a t i o n s e t f o r w h i c h a l l c l a s s i f i c a t i o n s i n t h a t s e t e q u a l z e r o ) . I f a l t e r n a t i v e 2 i s s e l e c t e d t h e u s e r may r e p o s i t i o n t h e s t a g e w i t h t h e j o y s t i c k and s u b s e q u e n t l y resume t h e s c a n , make a c l a s s i f i c a t i o n , o r r e s e t t h e s c a n c o o r d i n a t e s . I n t h e l a t t e r c a s e t h e s c a n c o o r d i n a t e s a r e n o t a d j u s t e d when t h e s t a g e i s moved by t h e u s e r ; t h a t i s , t h e c o m p u t e r t u r n s a b l i n d eye t o t h e m o t i o n o f t h e s t a g e . O p t i o n C : O p t i o n C computes t h e number o f o c c u r e n c e s o f e a c h c l a s s i f i c a t i o n i n e a c h c l a s s i f i c a t i o n s e t i n t h e d a t a f i l e " F i l e n a m e . L D A " . ( " F i l e n a m e " i s u s e r s u p p l i e d ) . A s e p a r a t e f r e q u e n c y i s c a l c u l a t e d f o r e a c h s e t o f c l a s s i f i c a t i o n s . The c o m p u t e r c a n be c o n s t r a i n e d t o i n c l u d e o n l y l o c a t i o n s o f a g i v e n c l a s s i f i c a t i o n i n a g i v e n c l a s s i f i c a t i o n s e t i n t h e f r e q u e n c y o f c l a s s i f i c a t i o n c a l c u l a t i o n . F o r e x a m p l e , i f d u r i n g t h e l o c a t i o n s c a n c e l l s a r e c l a s s i f i e d as 1 - ' h e a l t h y ' o r 2 - ' s i c k 1 and l a t e r t h e c e l l s a r e c l a s s i f i e d d u r i n g t h e c l a s s i f i c a t i o n s c a n a s 1 - ' s u r v i v o r ' o r 2 - ' k i l l e d ' , one c a n d e t e r m i n e t h e number o f h e a l t h y c e l l s t h a t s u r v i v e d and t h e number t h a t were k i l l e d as w e l l as t h e number o f s i c k c e l l s t h a t s u r v i v e d and t h e number t h a t were k i l l e d by s e t t i n g p a r a m e t e r F t o 1 u s i n g o p t i o n D t h e n by u s i n g o p t i o n C t o p rompt t h e c o m p u t e r f o r t h e r e s u l t s . C l a s s i f i c a t i o n S e t s 2 - 5 1 - 5 1 - 5 40 O p t i o n D: O p t i o n D i s u s e d t o l i s t a n d / o r r e d e f i n e t h e u s e r d e f i n a b l e p r o g r a m p a r a m e t e r s . These a r e l i s t e d b e l o w : PARAMETER A : SPEED B: PRINTOUT SET TO CLASSIFY RANGE 0 - 1 0 0 - 2 0 - 6 COLONY LOCATION CONSTRAINT PARAMETERS D: CLASSIF ICATION SET 0 COMMENT M o t o r s p e e d , 10 = maximum. 0 = No p r i n t o u t 1 = C l a s s i f i c a t i o n f r e q u e n c i e s o n l y . 2 = A l l p r i n t o u t 0 = No c l a s s i f i c a t i o n . 1 -5 = S e t t o c l a s s i f y . 6 = F i r s t s e t w i t h no c l a s s i f i -c a t i o n s . 0 = No c o n s t r a i n t 1 -5 = S e t c o n t a i n i n g c o n s t r a n t . C l a s s o f l o c a t i o n s t o be s c a n n e d . E : CLASSIF ICATION VALUE 0 - 5 CLASSIF ICATION FREQUENCY CONSTRAINT PARAMETERS F : CLASSIF ICATION SET 0 - 5 0 = No c o n s t r a i n t 1 -5 = S e t c o n t a i n i n g c o n s t r a i n t P a r a m e t e r s A , B , and C need no e x p l a n a t i o n . P a r a m e t e r s D and E a r e u s e d i f o n l y l o c a t i o n s w h i c h were p r e v i o u s l y c l a s s i f i e d w i t h a g i v e n v a l u e a r e t o be s c a n n e d ; f o r e x a m p l e , i f c e l l s were c l a s s i f i e d as 1 - ' s i c k ' o r 2 -• h e a l t h y ' when l o c a t e d w i t h "LDALOC" t h e u s e r c o u l d s e l e c t i v e l y s c a n t h e h e a l t h y c e l l s by s e t t i n g p a r a m e t e r D t o 1 , ( the c l a s s i f i c a t i o n s e t u s e d i n LDALOC) and p a r a m e t e r E t o 2 . The c o l o n y l o c a t i o n c o n s t r a i n t p a r a m e t e r s c a n be u s e d t o c l a s s i f y c e l l s n o t c l a s s i f i e d ( c l a s s i f i c a t i o n = 0 ) i n a g i v e n c l a s s i f i c a t i o n s e t , o r t o o n l y c l a s s i f y l o c a t i o n s o f a g i v e n c l a s s i f i c a t i o n w i t h i n a g i v e n c l a s s i f i c a t i o n s e t . P a r a m e t e r F i s u s e d i f a s e p a r a t e f r e q u e n c y o f c l a s s i f i c a t i o n c a l c u l a t i o n i s d e s i r e d f o r e a c h c l a s s i f i c a t i o n i n a g i v e n c l a s s i f i c a t i o n s e t as d e s c r i b e d p r e v i o u s l y . O p t i o n E : O p t i o n E i s u s e d t o s u b m i t t h e d a t a i n f i l e "LDAPRINT.OUT" t o t h e p r i n t e r . T h i s f i l e c o n t a i n s a l i s t i n g o f t h e p r i n t o u t r e q u e s t e d t h r o u g h o p t i o n D. A f t e r u s i n g o p t i o n D t h e c o n t e n t s o f "LDAPRINT.OUT" w i l l be e r a s e d and r e p l a c e d by new p r i n t o u t . 41 PURPOSE AND PROCEDURE 3 . 1 PURPOSE OF THE EXPERIMENTS. The p u r p o s e o f t h e s e e x p e r i m e n t s i s : 1 . To i m p r o v e t h e ' l o w dose a s s a y ' w h i c h i s u s e d t o measure c e l l s u r v i v a l a t s u r v i v a l l e v e l s g r e a t e r t h a n 50%, by r e d u c i n g e x p e r i m e n t e r s t r a i n , r e d u c i n g e x p e r i m e n t a l e r r o r , and i n c r e a s i n g p r o d u c t i v i t y . 2 . To m e a s u r e t h e e f f e c t o f m i s o n i d a z o l e o n t h e s u r v i v a l o f C h i n e s e h a m s t e r o v a r y (CHO) c e l l s e x p o s e d t o d o s e s o f r a d i a t i o n r a n g i n g f r o m 1 . 5 - 3 0 G r a y . 3 . To d e t e r m i n e t h e e f f e c t o f v a r y i n g c o n c e n t r a t i o n s o f m i s o n i d a z o l e on J t h e p a r a m e t e r s o f t h e l i n e a r - q u a d r a t i c m o d e l o f c e l l s u r v i v a l . S u r v i v a l i s a measure o f t h e p r o l i f e r a t i v e c a p a c i t y o f a c e l l . The o p e r a t i o n a l d e f i n i t i o n o f s u r v i v a l i s t h e r e l a t i v e number o f c e l l s w h i c h a r e a b l e t o d i v i d e t o f o r m c o l o n i e s o f more t h a n f i f t y c e l l s i n s e v e n d a y s (about 14 g e n e r a t i o n s ) . S u r v i v a l i s g e n e r a l l y n o r m a l i z e d t o t h e s u r v i v a l a t z e r o dose o f i o n i z i n g r a d i a t i o n , t h e p l a t i n g e f f i c i e n c y o f t h e c e l l l i n e . I t i s d i f f i c u l t t o measure s u r v i v a l l e v e l s g r e a t e r t h a n 0 . 5 0 w i t h t h e c o n v e n t i o n a l a s s a y as d e s c r i b e d by Puck and M a r c u s , 1956 ; c o n s e q u e n t l y t h e ' l o w d o s e a s s a y ' i s u s e d i n o u r l a b o r a t o r y f o r m e a s u r i n g s u r v i v a l l e v e l s f r o m a b o u t 0 . 5 0 - 0 . 9 0 w i t h u n c e r t a i n t i e s o f t h e o r d e r o f ±1%-±10% s t a n d a r d d e v i a t i o n . ( J . B r o s i n g PhD t h e s i s , 1 9 8 3 ) . T h i s a s s a y i s t i m e c o n s u m i n g and a s t r a i n on t h e e x p e r i m e n t e r . A m a j o r p o r t i o n o f t h i s t h e s i s i s d e v o t e d t o i m p r o v i n g t h e l o w dose a s s a y t o r e d u c e e x p e r i m e n t e r s t r a i n , r e d u c e e x p e r i m e n t a l e r r o r and t o s p e e d up t h e e x p e r i m e n t a l p r o c e d u r e . The s u r v i v a l o f mammal ian c e l l s a t l o w d o s e s o f i o n i z i n g i r r a d i a t i o n h a s i m p o r t a n t c l i n i c a l i m p l i c a t i o n s . P r e v i o u s i n v e s t i g a t o r s have s t u d i e d 42 t h e e f f e c t s o f t h e r a d i o s e n s i t i z e r m i s o n i d a z o l e on t h e s u r v i v a l o f mammal ian c e l l s s u b j e c t e d t o i o n i z i n g r a d i a t i o n u n d e r a n o x i c c o n d i t i o n s (Adams e t a l , 1976 ; M o o r e , 1976 ; M c N a l l y , 1 9 7 6 ) . Such i n v e s t i g a t i o n s have g e n e r a l l y b e e n c a r r i e d o u t a t f a i r l y h i g h c o n c e n t r a t i o n s o f m i s o n i d a z o l e ( 0 . 1 - 5 0 mM) and f a i r l y h i g h d o s e s o f r a d i a t i o n ( 5 - 3 0 G y ) . The r e s u l t s i n d i c a t e t h a t m i s o n i d a z o l e i s a good r a d i o s e n s i t i z e r u n d e r a n o x i c c o n d i t i o n s . The d o s e m o d i f y i n g f a c t o r (DMF) o f 15 mM m i s o n i d a z o l e i s a b o u t 2 . 5 f o r a d i l u t e s u s p e n s i o n o f a s y n c h r o n o u s CHO c e l l s . M i s o n i d a z o l e does n o t e f f e c t t h e c e l l s u r v i v a l t o any m e a s u r e a b l e e x t e n t u n d e r o x i c c o n d i t i o n s . ( e . g . Moore e t a l , 1976) Thus u n d e r t h e c o n d i t i o n s o f t h e s e e x p e r i m e n t s m i s o n i d a z o l e w i l l i n c r e a s e t h e k i l l o f a n o x i c c e l l s w i t h o u t f u r t h e r damag ing t h e o x i c c e l l s , an i m p o r t a n t c l i n i c a l a d v a n t a g e when t r e a t i n g t u m o u r s w h i c h c o n t a i n c e l l s w h i c h a r e i n an a n o x i c e n v i r o n m e n t . R e c e n t f i n d i n g s b y P a l c i c e t a l (1982) show a s i g n i f i c a n t r e d u c t i o n o f t h e OER o f CHO c e l l s i r r a d i a t e d t o d o s e s o f l e s s t h a n 2 Gy . O t h e r l i t e r a t u r e s u p p o r t s t h e h y p o t h e s i s t h a t m i s o n i d a z o l e and o x y g e n a c t a s r a d i o s e n s i t i z e r s by a s i m i l a r m e c h a n i s m . (Adams, 1973 ; Wardman, 1977) I f t h i s i s so t h e r e may w e l l be a r e d u c t i o n o f t h e m i s o n i d a z o l e DMF m e a s u r e d a t l o w d o s e s o f i r r a d i a t i o n ( i . e . , a t h i g h s u r v i v a l l e v e l s ) , a n a l o g o u s t o t h e r e d u c t i o n o f t h e o x y g e n e f f e c t ( f i g u r e 2 ) . A t y p i c a l tumour t r e a t m e n t t o d a y w o u l d i n v o l v e 20 f r a c t i o n s o f a b o u t 2 Gy e a c h o v e r t h e c o u r s e o f s e v e r a l w e e k s . M i s o n i d a z o l e c o n c e n t r a t i o n s a r e g e n e r a l l y l e s s t h a n 0 . 1 mM i n t h e c l i n i c . I t i s i m p o r t a n t t o measure what e f f e c t m i s o n i d a z o l e h a s , i f any on t h e s u r v i v a l o f mammalian c e l l s a t c l i n i c a l d o s e s o f r a d i a t i o n and c o n c e n t r a t i o n s o f m i s o n i d a z o l e . I n t h i s t h e s i s t h e e f f e c t o f m i s o n i d a z o l e on t h e s u r v i v a l o f a n o x i c CHO c e l l s s u b j e c t e d t o 270 kVP X - r a y s i s m e a s u r e d 43 o v e r a w i d e r a n g e o f s e n s i t i z e r c o n c e n t r a t i o n ( 0 - 4 0 mM) and r a d i a t i o n dose ( 0 - 3 0 Gray ) . C e l l s were k e p t on i c e d u r i n g t h e t i m e o f t r e a t m e n t w i t h m i s o n i d a z o l e i n o r d e r t o m i n i m i z e c y t o t o x i c e f f e c t s . The s u r v i v a l o f mammalian c e l l s a t l o w d o s e s o f i o n i z i n g r a d i a t i o n h a s i m p o r t a n t r a d i o b i o l o g i c a l i m p l i c a t i o n s . S u r v i v a l m o d e l s p r e d i c t t h e shape o f t h e c e l l s u r v i v a l c u r v e a t l o w d o s e o f r a d i a t i o n ( 0 - 5 Gy) b a s e d o n measurements a t h i g h d o s e s o f r a d i a t i o n ( 5 - 3 0 G y ) . Such m o d e l s i n c l u d e m u l t i t a r g e t t h e o r y (Puck and M a r c u s , 1956 ; B e n d e r and G o o c h , 1962) , t h e l i n e a r - q u a d r a t i c m o d e l ( S i n c l a i r , 1966 ; K e l l e r e r and R o s s i , 1978; C h a d w i c k and L e e n h o u t s , 1 9 8 1 ) , and t h e r e p a i r - m i s r e p a i r m o d e l ( C . T o b i a s , 1 9 8 0 ) . B e c a u s e o f t h e i n h e r e n t u n c e r t a i n t y i n b i o l o g i c a l d a t a (due t o t h e l a r g e number o f h i g h l y c o r r e l a t e d p a r a m e t e r s s u c h as c e l l e n v i r o n m e n t , c e l l h i s t o r y , e t c , w h i c h c a n e f f e c t c e l l s u r v i v a l ) i t i s d i f f i c u l t ( pe rhaps i m p o s s i b l e ) t o d i s t i n g u i s h b e t w e e n t h e s e m o d e l s on t h e b a s i s o f c e l l s u r v i v a l d a t a ( e . g . B e n d e r and G o o c h , 1 9 6 2 ) . The g o a l o f a mode l i s t o u s e a minimum o f a c c e p t a b l e h y p o t h e s e s t o a d e q u a t e l y d e s c r i b e a s e t o f e x p e r i m e n t a l s y s t e m s and t o p r e d i c t t h e e f f e c t o f c h a n g e s i n b a s i c p a r a m e t e r s w i t h i n t h e s e s y s t e m s . I n t h i s way m o d e l s c o n t r i b u t e t o o u r g e n e r a l u n d e r s t a n d i n g o f t h e f i e l d o f s t u d y and p r o v i d e f o r d i r e c t i o n i n o u r r e s e a r c h . I n o r d e r t o f i n d a s u i t a b l e m o d e l o r t o d i s t i n g u i s h b e t w e e n t h e p r a c t i c a l i t y o f e x i s t i n g m o d e l s , p r e d i c t i o n s made by t h e s e m o d e l s must be s u b j e c t e d t o e x p e r i m e n t a l v e r i f i c a t i o n . My c o n t r i b u t i o n t o t h e d e v e l o p m e n t o f r a d i o b i o l o g i c a l m o d e l s was t o d e t e r m i n e how some d o s e i n d e p e n d e n t p a r a m e t e r s v a r y w i t h v a r y i n g m i s o n i d a z o l e c o n c e n t r a t i o n . Two s e t s o f e x p e r i m e n t s were p e r f o r m e d t o measure t h e e f f e c t o f m i s o n i d a z o l e on c e l l s u r v i v a l . E x p e r i m e n t s e t A showed t h e g e n e r a l t r e n d o f t h e dependence o f s u r v i v a l on m i s o n i d a z o l e c o n c e n t r a t i o n up t o t h e l e v e l 44 a t w h i c h no f u r t h e r i n c r e a s e i n c e l l k i l l i s o b s e r v e d . A n a l y s i s o f t h e d a t a f r o m e x p e r i m e n t s e t A i n d i c a t e d t h a t t h e l o g o f t h e s u r v i v a l was p r o p o r t i o n a l t o t h e X - r a y dose and t o t h e s q u a r e o f t h e X - r a y dose as f o l l o w s : 2 l o g ^ Q S = - a D - b D E q u a t i o n 3 S = c e l l s u r v i v a l . D=X - ray d o s e . a = a l p h a , a m i s o n i d a z o l e d e p e n d e n t p a r a m e t e r . b = b e t a , a m i s o n i d a z o l e d e p e n d e n t p a r a m e t e r . The c h o i c e o f e q u a t i o n 3 f o r f i t t i n g t h e d a t a does n o t c o n s t i t u t e a b i a s t o w a r d t h e m o l e c u l a r t h e o r y o f c e l l s u r v i v a l o r any o t h e r m o d e l w h i c h i s c o n s i s t e n t w i t h t h i s s u r v i v a l e q u a t i o n . T h i s e q u a t i o n was c h o s e n a s one o f t h e s i m p l e s t w h i c h c a n a d e q u a t e l y f i t t h e d a t a . A l l m o d e l s c a n be w r i t t e n a s an i n f i n i t e p o l y n o m i a l i n d o s e . F o r t h e g r e a t m a j o r i t y o f e x p e r i m e n t a l d a t a r e p o r t e d i n t h e l i t e r a t u r e t h e l i n e a r and q u a d r a t i c t e r m s a r e s u f f i c i e n t t o f i t s u r v i v a l d a t a o v e r t h e r a n g e o f d o s e s u s e d . F o r e x p e r i m e n t s e t A t h e c o e f f i c i e n t o f t h e l i n e a r component o f t h e d o s e , a l p h a , d i d n o t v a r y s i g n i f i c a n t l y w i t h m i s o n i d a z o l e c o n c e n t r a t i o n b u t r e m a i n e d c o n s t a n t a t 0.042±0.010 G r a y 1 . The c o e f f i c i e n t o f t h e q u a d r a t i c component o f t h e d o s e , b e t a , i n c r e a s e d f r o m a p p r o x i m a t e l y - 2 - 2 0 . 0 0 2 3 G r a y t o 0 . 0 0 6 5 G r a y i n t h e m i s o n i d a z o l e c o n c e n t r a t i o n r a n g e o f O. lmM t o 4 0 . 0 mM. T h e s e p r e l i m i n a r y r e s u l t s p r o v i d e d t h e m o t i v a t i o n f o r e x p e r i m e n t s e t B. T h i s s e t o f e x p e r i m e n t s was d e s i g n e d t o m i n i m i z e e x p e r i m e n t a l u n c e r t a i n t y and t o more c a r e f u l l y d e f i n e a l p h a i n t h e m i s o n i d a z o l e c o n c e n t r a t i o n r a n g e i n w h i c h b e t a was c h a n g i n g most q u i c k l y . The a u t o m a t e d l o w d o s e a s s a y s y s t e m (ALDAS) was u s e d t o h e l p i m p r o v e e x p e r i m e n t a l s t a t i s t i c s and r e d u c e e x p e r i m e n t a l u n c e r t a i n t y by a l l o w i n g f o r 45 i m p r o v e d e x p e r i m e n t a l d e s i g n and by r e d u c i n g t h e s t r a i n on t h e e x p e r i m e n t e r i n l o c a t i n g and c l a s s i f y i n g c e l l s . The r e s u l t s o f e x p e r i m e n t s e t B show e q u a t i o n 3 t o be a r e a s o n a b l e f i t t o t h e d a t a . A l p h a r e m a i n s c o n s t a n t a t 0.039±0.010 G r a y and b e t a i n c r e a s e s f r o m - 2 - 2 0.0019±0.0002 G r a y t o 0 . 0 1 2 1 0 . 0 0 2 G r a y i n t h e m i s o n i d a z o l e c o n c e n t r a t i o n r a n g e o f 0 . 1 mM t o 5 . 0 mM. 3 . 2 EXPERIMENT SET A 3 . 2 . 1 P u r p o s e and p r o c e d u r e . The p u r p o s e o f t h i s s e t o f e x p e r i m e n t s w a s : 1 . To d e v e l o p an e x p e r i m e n t a l p r o c e d u r e f o r m e a s u r i n g t h e e f f e c t s o f v a r y i n g c o n c e n t r a t i o n s o f m i s o n i d a z o l e on c e l l s u r v i v a l when c e l l s a r e e x p o s e d t o X - r a y s . 2 . To measure t h e dependence o f c e l l s u r v i v a l on m i s o n i d a z o l e c o n c e n t r a t i o n . 3 . To e s t i m a t e t h e l i n e a r and q u a d r a t i c c o e f f i c i e n t s a l p h a and b e t a o f t h e s u r v i v a l c u r v e d e s c r i b e d b y e q u a t i o n 3 and t h e i r dependence on t h e m i s o n i d a z o l e c o n c e n t r a t i o n . CHO c e l l s f r o m s p i n n e r f l a s k s ( s u s p e n s i o n c u l t u r e ) were d i l u t e d t o 5 1X10 c e l l s / m l e l e v e n h o u r s p r i o r t o i r r a d i a t i o n . The c e l l s have a d o u b l i n g t i m e o f a p p r o x i m a t e l y 14 h o u r s , and t h e y r e m a i n e d e x p o n e n t i a l l y g r o w i n g t h r o u g h o u t t h e t i m e c o u r s e o f t h e e x p e r i m e n t . 5 - 4 0 mM m i s o n i d a z o l e was p r e p a r e d i n b u l k f r o m w h i c h t h e r e q u i r e d m i s o n i d a z o l e c o n c e n t r a t i o n s were p r e p a r e d by f u r t h e r d i l u t i o n w i t h medium. B e f o r e i r r a d i a t i o n 2 . 8 X 1 0 ^ c e l l s were removed f r o m t h e s u s p e n s i o n , c e n t r i f u g e d f o r s i x m i n u t e s a t 600 r . p . m . and r e s u s p e n d e d i n 1 m l a l p h a medium w i t h f e t a l c a l f serum (FCS) and no NaHCO^, v o r t e x e d , and p i p e t t e d i n t o 19 ml o f a l p h a medium w i t h FCS and no NaHCO^ w i t h m i s o n i d a z o l e added as r e q u i r e d . C e l l s were t h e n p l a c e d i n i r r a d i a t i o n v e s s e l s on i c e and p u r i f i e d n i t r o g e n gas (N^, l e s s t h a n 5 ppm 0 o ) was f l o w e d i n t o t h e v e s s e l o v e r t h e medium f o r 46 a p e r i o d o f 4 5 - 9 0 m i n u t e s . The s o u r c e o f i r r a d i a t i o n u s e d was a P i c k e r X - r a y m a c h i n e (270 k V P , 0 . 4 0 - 1 . 6 0 G y / m i n ) . The d o s e r a t e was c a l c u l a t e d u s i n g f e r r o u s s u l p h a t e d o s i m e t r y t e c h n i q u e s ( F r i c k e and H a r t , i n R a d i a t i o n D o s i m e t r y , v o l . I I , page 167 , A t t i x and T o e s c h e d , 1 9 6 6 ) . Samples were i r r a d i a t e d on i c e w i t h N 2 g a s f l o w i n g c o n s t a n t l y t h r o u g h t h e i r r a d i a t i o n v e s s e l . D u r i n g t h e c o u r s e o f i r r a d i a t i o n , a l i q u o t s o f 0 . 5 - 3 . 0 m l were removed i n t o 9 . 5 m l a l p h a medium (no FCS) and s t o r e d on i c e f o r up t o one h o u r . I n some o f t h e e x p e r i m e n t s c e l l s were c e n t r i f u g e d f o r s i x m i n u t e s a t 600 r . p . m . p r i o r t o p l a t i n g t o remove m i s o n i d a z o l e . C e l l s e x p o s e d t o l o w d o s e s o f i r r a d i a t i o n were p l a t e d u s i n g t h e l o w dose a s s a y , and t h o s e e x p o s e d t o t h e h i g h e r d o s e s o f r a d i a t i o n were p l a t e d u s i n g t h e c o n v e n t i o n a l a s s a y . C e l l p l a t i n g f o r t h e l o w d o s e and c o n v e n t i o n a l a s s a y t o o k 1 . 5 - 2 h o u r s . An a v e r a g e o f a b o u t 150 c e l l s were p l a t e d p e r m i c r o t e s t p l a t e f o r t h e l o w dose a s s a y . Up t o f o u r v e s s e l s w i t h d i f f e r e n t m i s o n i d a z o l e c o n c e n t r a t i o n s were i r r a d i a t e d d u r i n g t h e c o u r s e o f s i x h o u r s . 3 . 2 . 2 C h o o s i n g b e t w e e n t h e l o w d o s e and c o n v e n t i o n a l a s s a y . The low dose a s s a y i s a more s u i t a b l e means o f a s s a y i n g c e l l p r o l i f e r a t i v e c a p a c i t y t h a n t h e c o n v e n t i o n a l a s s a y a t l o w d o s e s . The l o w dose a s s a y r e q u i r e s a p p r o x i m a t e l y 2 0 - 3 0 m i n u t e s t o p l a t e , l o c a t e and c l a s s i f y 1 0 0 - 2 0 0 c e l l s , ( b e f o r e a u t o m a t i o n , a p p r o x i m a t e l y 45 m i n u t e s ) . The c o n v e n t i o n a l a s s a y r e q u i r e s a b o u t f i v e m i n u t e s t o c o u n t , p l a t e , s t a i n and c o u n t two t o t h r e e p e t r i d i s h e s w i t h 1 0 0 - 2 0 0 c o l o n i e s e a c h . Thus t h e l o w dose a s s a y i s more s u i t a b l e o n l y when i t i m p r o v e s m a r k e d l y upon t h e u n c e r t a i n t y o f t h e c o n v e n t i o n a l a s s a y . The a c c u r a c y o f t h e c o n v e n t i o n a l a s s a y i s l i m i t e d p r i m a r i l y by t h e u n c e r t a i n t y i n t h e number o f c e l l s p l a t e d . T h i s c a n be r e d u c e d by e i t h e r p l a t i n g more c e l l s ( i f t h e c e l l s a r e n o r m a l l y d i s t r i b u t e d t h r o u g h o u t t h e i r r a d i a t e d s a m p l e s one needs t o p l a t e a b o u t f o u r t i m e s as 47 many c e l l s t o r e d u c e t h e u n c e r t a i n t y by a f a c t o r o f two) o r by a c c u r a t e l y c o u n t i n g t h e number o f c e l l s p l a t e d as i n t h e l o w dose a s s a y . I n s e c t i o n 4 . 1 . 3 i t i s shown t h a t i n t h e c a s e o f 50% s u r v i v a l o f p l a t e d c e l l s i t w o u l d be n e c e s s a r y t o p l a t e a p p r o x i m a t e l y 1500 c e l l s , s u r v i v o r s and k i l l e d , ( a p p r o x i m a t e l y s e v e n p e t r i d i s h e s o f 100 s u r v i v o r s e a c h ) , t o a c h i e v e t h e same a c c u r a c y as w i t h t h e l o w d o s e a s s a y when 200 c e l l s a r e l o c a t e d and c l a s s i f i e d . T h i s a n a l y s i s t a k e s i n t o a c c o u n t t h e p l a t i n g e f f i c i e n c i e s o f t h e s e a s s a y s . I t i s much e a s i e r t o p l a t e and s u b s e q u e n t l y h a n d l e one 2 v e s s e l ( m i c r o t e s t p l a t e , 25cm f l a s k , e t c . ) t h a n t e n p e t r i d i s h e s ( w h i c h must be c o u n t e d , d i l u t e d , p l a t e d , e t c . i n d e p e n d e n t l y t o r e d u c e s y s t e m a t i c e r r o r s ) . I n p r a c t i c e we u s e t h e l o w d o s e a s s a y t o measure s u r v i v a l o f c e l l s a t l e v e l s g r e a t e r t h a n 50%. 3 . 2 . 3 S u r v i v a l d a t a . S u r v i v a l d a t a a r e o b t a i n e d w i t h e i t h e r t h e l o w dose a s s a y o r t h e c o n v e n t i o n a l a s s a y . C a l c u l a t i o n s o f s u r v i v a l a r e h a n d l e d d i f f e r e n t l y f o r t h e s e two a s s a y s and a r e c a r r i e d o u t as f o l l o w s : 1 . F o r t h e l o w d o s e a s s a y s u r v i v a l i s c a l c u l a t e d as t h e number o f c e l l s c l a s s i f i e d as s u r v i v o r s on t h e c l a s s i f i c a t i o n day d i v i d e d by t h e t o t a l number o f c e l l s l o c a t e d on t h e l o c a t i o n d a y , c o r r e c t e d f o r p o s s i b l e l o c a t i o n e r r o r s , a c c o r d i n g t o e q u a t i o n 2 . The s u r v i v a l i s n o r m a l i z e d t o t h e a v e r a g e s u r v i v a l a t z e r o dose f o r t h e e x p e r i m e n t . The s t a n d a r d d e v i a t i o n o f t h e s u r v i v a l i s e s t i m a t e d a c c o r d i n g t o e q u a t i o n 5 . 2 . F o r t h e c o n v e n t i o n a l a s s a y s u r v i v a l i s c a l c u l a t e d as t h e number o f c o l o n i e s c o u n t e d a f t e r s t a i n i n g d i v i d e d by t h e e s t i m a t e d number o f c e l l s p l a t e d , a c c o r d i n g t o e q u a t i o n 1 . The s u r v i v a l i s n o r m a l i z e d t o t h e a v e r a g e s u r v i v a l a t z e r o d o s e f o r t h e e x p e r i m e n t . The s t a n d a r d d e v i a t i o n o f t h e s u r v i v a l i s e s t i m a t e d a c c o r d i n g t o e q u a t i o n 9 , s e c t i o n 4 . 1 . 3 . The c h i - s q u a r e d t e s t was u s e d t o e s t i m a t e t h e most l i k e l y v a l u e s o f 48 t h e p a r a m e t e r s a l p h a and b e t a o f t h e l i n e a r - q u a d r a t i c e q u a t i o n ( e q u a t i o n 3) as w e l l as t h e i r 70% c o n f i d e n c e i n t e r v a l s . D e t a i l s o f t h e a n a l y s i s and t h e r e s u l t s a r e p r e s e n t e d i n s e c t i o n 4 . 3 . 3 EXPERIMENT SET B . 3 . 3 . 1 M o t i v a t i o n f o r an i m p r o v e d l o w d o s e a s s a y p r o c e d u r e . The low d o s e a s s a y d e s c r i b e d i n s e c t i o n f o u r i s u s e f u l f o r m e a s u r i n g c e l l s u r v i v a l a t s u r v i v a l l e v e l s o f 0 . 5 0 o r g r e a t e r . The minimum e q u i p m e n t needed t o p e r f o r m t h i s a s s a y i s a m i c r o s c o p e w i t h a m a n u a l l y d r i v e n s t a g e . D a t a f o r e x p e r i m e n t s e t A was o b t a i n e d u s i n g s u c h e q u i p m e n t . The a s s a y i s a s t r a i n on t h e e x p e r i m e n t e r , m a i n l y due t o t h e c o n c e n t r a t i o n r e q u i r e d t o l o c a t e a s m a l l c e l l o f a b o u t 10 um d i a m e t e r i n a l a r g e f i e l d o f a b o u t 2 mm d i a m e t e r and t o r e f o c u s t h e e y e s on g r a p h p a p e r t o m a n u a l l y r e c o r d t h e l o c a t i o n o f t h e c e l l . T h i s p r o c e d u r e must be p e r f o r m e d w i t h s u f f i c i e n t s p e e d and a c c u r a c y t o d e r i v e s t a t i s t i c a l b e n e f i t s o v e r t h e c o n v e n t i o n a l s u r v i v a l a s s a y . T h e r e i s t h e d a n g e r o f a t i r e d e x p e r i m e n t e r m i s s i n g c e l l s i n f a v o u r o f h e a l t h y , d i v i d i n g c e l l s and t h e r e b y a d d i n g a s y s t e m a t i c e r r o r t o t h e s u r v i v a l r e s u l t s . The a u t o m a t e d l o w d o s e a s s a y s y s t e m (ALDAS) c o n s i s t s o f a c o m p u t e r -c o n t r o l l e d , s t e p p i n g - m o t o r - d r i v e n m i c r o s c o p e s t a g e . The s t a g e m o t i o n i s c o n t r o l l e d by t h e o b s e r v e r t h r o u g h a j o y s t i c k . ALDAS o f f e r s many b e n e f i t s o v e r t h e m a n u a l s y s t e m . The c o m p u t e r t a k e s o v e r t h e t a s k o f k e e p i n g t r a c k o f t h e s t a g e l o c a t i o n , t h u s a h i g h e r o b j e c t i v e power c a n be u s e d s o t h a t c e l l s c a n be r e c o g n i z e d more e a s i l y . T h i s r e s u l t s i n f e w e r e r r o r s i n c e l l r e c o g n i t i o n and l e s s e x p e r i m e n t e r s t r a i n . The c o m p u t e r a l s o t a k e s o v e r t h e t a s k o f r e c o r d i n g t h e c e l l l o c a t i o n s , t h u s a r e f e r e n c e g r i d on t h e v e s s e l i n w h i c h t h e c e l l s a r e p l a t e d i s n o t r e q u i r e d and c e l l s c a n be p l a t e d i n 25 49 cm f l a s k s i n s t e a d o f m i c r o t e s t p l a t e s . P l a t i n g and l o c a t i n g c e l l s i n f l a s k s i s much e a s i e r and f a s t e r . C e l l s a r e more l i k e l y t o c o n t a c t a w e l l w a l l when p l a t e d i n m i c r o t e s t p l a t e s , m a k i n g them more d i f f i c u l t t o l o c a t e and c l a s s i f y . P h a s e o p t i c s c a n n o t be u s e d e f f e c t i v e l y w i t h m i c r o t e s t p l a t e s . The a u t o m a t e d s y s t e m g r e a t l y i m p r o v e s t h e l o c a t i o n a c c u r a c y t o b e t t e r t h a n 100 um o r 5 - 1 0 c e l l d i a m e t e r s so t h a t c e l l s c a n be p l a t e d more c l o s e l y and s t i l l be e a s y t o i d e n t i f y on t h e c l a s s i f i c a t i o n d a y . P l a t i n g c e l l s a t , a h i g h e r d e n s i t y a l s o makes them e a s i e r t o l o c a t e . The f o l l o w i n g i s a summary o f t h e l o w d o s e a s s a y p r o c e d u r e s u s e d b e f o r e ALDAS ( e x p e r i m e n t s e t A) and a f t e r ALDAS ( e x p e r i m e n t s e t B ) : Low Dose As B e f o r e ALDAS 1 . C e l l s p l a t e d i n m i c r o t e s t p l a t e s . 2 . L o c a t e c e l l s i n t h e m i c r o t e s t p l a t e s by m a n u a l l y m o v i n g t h e m i c r o s c o p e s t a g e . C e l l s l o c a t e d 2 - 3 d a y s f o l l o w i n g i r r a d i a t i o n . 3 . Upon l o c a t i n g e a c h c e l l , r e c o r d e a c h c e l l l o c a t i o n on g r a p h p a p e r . R e p e a t a b i l i t y a b o u t 5 0 0 - 1 0 0 0 um. 4 . 6 - 8 d a y s f o l l o w i n g i r r a d i a t i o n r e t u r n t o t h e a p p r o x i m a t e l o c a -t i o n o f e a c h l o c a t e d c e l l and c l a s s i f y . 5 . Add up t h e number o f c e l l s w i t h e a c h g i v e n c l a s s i f i c a t i o n and c a l c u l a t e s u r v i v a l . p r o c e d u r e A f t e r ALDAS 2 1 . C e l l s p l a t e d i n 25cm a r e a f l a s k s . 2 . L o c a t e c e l l s i n t h e f l a s k s u s i n g t h e ALDAS j o y s t i c k t o move t h e s t a g e . C e l l s l o c a t e d 2 - 3 d a y s f o l l o w i n g i r r a d i a t i o n . 3 . Upon l o c a t i n g e a c h c e l l , s i g n a l t h e c o m p u t e r t o r e c o r d t h e c e l l l o c a t i o n . R e p e a t a b i l i t y o f 1 0 - 1 0 0 um. 4 . 6 - 8 d a y s f o l l o w i n g i r r a d i a t i o n u s e t h e c o m p u t e r t o r e t u r n t o t h e c e l l w i t h i n 1 0 - 1 0 0 um o f e a c h l o c a t e d c e l l and c l a s s i f y . 5 . Computer c o m p i l e s t h e d a t a and c a l c u l a t e s s u r v i v a l . A d d i t i o n a l a d v a n t a g e s o f ALDAS i n c l u d e s p e e d and a c c u r a c y i n d a t a p r o c e s s i n g and t h e a b i l i t y t o p e r f o r m new e x p e r i m e n t s i n v o l v i n g t h e c h a n g e s i n c e l l m o r p h o l o g y as a f u n c t i o n o f t i m e , r a d i a t i o n d o s e , and s e n s i t i z e r c o n c e n t r a t i o n . The l a t t e r w o u l d i n v o l v e t h e u s e o f a t e l e v i s i o n camera and r e c o r d e r . 50 3 . 3 . 2 ALDAS and t h e l o w d o s e a s s a y . T h i s s e c t i o n d e s c r i b e s how ALDAS was u s e d i n e x p e r i m e n t s e t B t o measure t h e s u r v i v a l o f c e l l s w i t h t h e l o w dose a s s a y . C e l l s t o be a s s a y e d were p l a t e d i n 25 c m 2 c u l t u r e f l a s k s ( f i g u r e 5 ) . B e f o r e p l a t i n g , t h e s c a n b o u n d a r i e s and r e f e r e n c e p o i n t o f t h e f l a s k were d e f i n e d as f o l l o w s : 1 . An empty f l a s k was s e t on t h e s t a g e on a h o l d e r s p e c i f i c a l l y d e s i g n e d f o r t h e f l a s k . The h o l d e r was d e s i g n e d s u c h t h a t t h e f l a s k was h e l d i n a s p e c i f i c o r i e n t a t i o n as r i g i d l y as p o s s i b l e and t h e o b j e c t i v e c o u l d s c a n t h e e n t i r e a r e a o f t h e f l a s k w i t h o u t h i t t i n g t h e h o l d e r . The s i d e s o f t h e f l a s k were o r i e n t e d p a r a l l e l t o t h e s t a g e l e a d s c r e w s . 2 . The LOCATION SCAN MENU o p t i o n C was u s e d . F i r s t two d i a g o n a l l y o p p o s i t e c o r n e r s o f t h e f l a s k were l o c a t e d i n o r d e r t o d e f i n e t h e r e c t a n g u l a r b o u n d a r y o f t h e f l a s k . T h i s d e f i n e s t h e l i m i t s o f s t a g e movement d u r i n g t h e l o c a t i o n and c l a s s i f i c a t i o n s c a n s . N e x t t h e r e f e r e n c e p o s i t i o n on t h e f l a s k ( f i g u r e 5) was l o c a t e d . No s c a n p o i n t s were i n p u t . These were p r o v i d e d by t h e s o f t w a r e . S i n c e a l l t h e f l a s k s u s e d f o r t h e l o w d o s e a s s a y f o r e x p e r i m e n t s e t B were e s s e n t i a l l y i d e n t i c a l , t h i s b o u n d a r y and r e f e r e n c e p o i n t l o c a t i o n was n e v e r r e p e a t e d . The same c o o r d i n a t e s were u s e d f o r a l l o f t h e s a m p l e s . A f t e r t h e s c a n b o u n d a r i e s and r e f e r e n c e p o s i t i o n were d e f i n e d t h e e x p e r i m e n t s c o u l d go a h e a d . I r r a d i a t e d c e l l s were p l a t e d i n t h e 25 c m 2 c u l t u r e f l a s k s and i n c u b a t e d f o r two t o t h r e e d a y s . The s m a l l c o l o n i e s o f a b o u t 1 -20 c e l l s ( c l o n e s o f t h e o r i g i n a l p l a t e d c e l l s ) were t h e n l o c a t e d w i t h t h e h e l p o f ALDAS. I n t r i a l e x p e r i m e n t s w i t h ALDAS i t was o b s e r v e d ( q u a l i t a t i v e l y ) t h a t t h e p l a t i n g e f f i c i e n c y i n c r e a s e s w i t h t h e number o f d a y s b e t w e e n i r r a d i a t i o n and l o c a t i o n . Thus i t was i m p o r t a n t t o l o c a t e a l l s a m p l e s w h i c h were i r r a d i a t e d w i t h t h e same c o n c e n t r a t i o n o f m i s o n i d a z o l e on t h e same day so t h a t t h e y c o u l d a l l be c o r r e c t e d by t h e same p l a t i n g 51 e f f i c i e n c y as m e a s u r e d by t h e z e r o i r r a d i a t i o n d o s e s a m p l e . C e l l s were l o c a t e d as f o l l o w s : 1 . The f l a s k was mounted on t h e m i c r o s c o p e s t a g e i n t h e same o r i e n t a t i o n as t h e f l a s k u s e d t o d e f i n e t h e s c a n b o u n d a r i e s and r e f e r e n c e p o s i t i o n . 2 . LDALOC LOCATION SCAN MENU o p t i o n A , ' S t a r t r e g u l a r s c a n ' , was u s e d . The name o f t h e d a t a s e t t o w h i c h r e c o r d e d l o c a t i o n s were t o be o u t p u t was e n t e r e d . 3 . The f l a s k r e f e r e n c e p o i n t was l o c a t e d u s i n g t h e j o y s t i c k . 4 . A f t e r l o c a t i n g t h e r e f e r e n c e p o s i t i o n t h e s t a g e was c o n t r o l l e d b y t h e a u t o m a t i c s c a n mode; t h a t i s , t h e d i r e c t i o n o f t h e s t a g e was s o f t w a r e c o n t r o l l e d , and t h e s p e e d o f t h e s t a g e was c o n t r o l l e d t h r o u g h t h e j o y s t i c k . The j o y s t i c k was u s e d t o c o n t r o l t h e s p e e d o f t h e s t a g e t o t h e f i r s t s o f t w a r e g e n e r a t e d a u t o m a t i c s c a n p o i n t . The e x p e r i m e n t e r now b e g a n t o l o c a t e t h e c e l l s i n t h e f l a s k . 5 . The j o y s t i c k was u s e d t o c o n t r o l t h e s c a n s p e e d t o t h e n e x t a u t o m a t e d s c a n p o i n t . I f a c e l l was o b s e r v e d , b u t t o n one on t h e j o y s t i c k c o n s o l e was p r e s s e d t o s t o p t h e a u t o m a t e d s c a n . The s t a g e was t h e n c o n t r o l l e d by t h e m a n u a l s c a n mode i n w h i c h t h e j o y s t i c k c o n t r o l s b o t h d i r e c t i o n and s p e e d o f t h e s t a g e . The j o y s t i c k was u s e d t o c e n t r e t h e c e l l u n d e r t h e m i c r o s c o p e c r o s s h a i r s and b u t t o n one on t h e j o y s t i c k c o n s o l e was p r e s s e d t o r e c o r d t h e l o c a t i o n . The s t a g e was t h e n c o n t r o l l e d b y t h e a u t o m a t i c s c a n and t h i s s t e p was r e p e a t e d . A f t e r t h e c e l l s were l o c a t e d i n e a c h c u l t u r e f l a s k t h e y were i n c u b a t e d a t 37°C w i t h t h e l i d o f t h e f l a s k t i g h t l y c l o s e d . T h e r e was s u f f i c i e n t medium i n e a c h f l a s k so t h a t C 0 2 f l o w was n o t needed t o m a i n t a i n t h e c o r r e c t pH o f 7 . 4 . 52 S i x t o s e v e n d a y s a f t e r i r r a d i a t i o n t h e f l a s k s were removed f r o m t h e i n c u b a t o r and t h e c e l l s were c l a s s i f i e d u s i n g p r o g r a m LDACLASS as f o l l o w s : 1 . LDACLASS CLASSIF ICATION SCAN MENU o p t i o n A , ' S t a r t s c a n ' , was u s e d . 2 . The name o f t h e d a t a s e t w h i c h c o n t a i n s t h e l o c a t i o n s o f t h e c e l l s i n t h e f l a s k t o be c l a s s i f i e d was e n t e r e d . 3 . The f l a s k r e f e r e n c e p o i n t was l o c a t e d u s i n g t h e j o y s t i c k . 4 . The s t a g e was moved a u t o m a t i c a l l y t o t h e f i r s t c e l l l o c a t i o n . The c o l o n y a t t h i s l o c a t i o n was e i t h e r m o n i t o r e d t h r o u g h t h e m i c r o s c o p e e y e p i e c e o r on t h e TV. 5 . B u t t o n one o f t h e j o y s t i c k c o n s o l e was p r e s s e d t h e n t h e c o l o n y c l a s s i f i c a t i o n was e n t e r e d . The f o l l o w i n g c l a s s i f i c a t i o n s were u s e d : 1 - s u r v i v o r , 2 - k i l l e d , 3 - c o i n c i d e n c e , and 4 - p o s s i b l e e r r o r . (See s e c t i o n 2 . 3 f o r a c o m p l e t e d e s c r i p t i o n o f t h e s e c l a s s i f i c a t i o n s . ) The s t a g e was moved a u t o m a t i c a l l y t o t h e n e x t c e l l l o c a t i o n and t h i s s t e p was r e p e a t e d . A f t e r a l l o f t h e c e l l s were c l a s s i f i e d t h e s t a g e was a u t o m a t i c a l l y r e t u r n e d t o t h e r e f e r e n c e p o s t i t i o n and t h e c e l l l o c a t i o n s a l o n g w i t h t h e c l a s s i f i c a t i o n s were o u t p u t t o t h e d a t a s e t named by t h e u s e r . A f t e r t h e c e l l s were c l a s s i f i e d t h e c o m p u t e r was u s e d t o d e t e r m i n e t h e number o f c o l o n i e s w i t h e a c h g i v e n c l a s s i f i c a t i o n . F o r e x p e r i m e n t s e t B g e n e r a l l y no c l a s s i f i c a t i o n was made d u r i n g t h e l o c a t i o n s c a n and o n l y one c l a s s i f i c a t i o n p e r c o l o n y ( r e c o r d e d l o c a t i o n ) was made u s i n g t h e c l a s s i f i c a t i o n s c a n . Thus t h e d e f a u l t p r o g r a m p a r a m e t e r s were s u f f i c i e n t t o d e t e r m i n e t h e c l a s s i f i c a t i o n f r e q u e n c y . The r e s u l t s were o b t a i n e d as f o l l o w s : 1 . LDACLASS CLASSIF ICATION SCAN MENU o p t i o n C was u s e d . 53 2 . The name o f t h e d a t a s e t f o r w h i c h t o d e t e r m i n e t h e c l a s s i f i c a t i o n f r e q u e n c y was e n t e r e d . 3 . The c o m p u t e r o u t p u t t h e number o f t i m e s e a c h c l a s s i f i c a t i o n o c c u r e d i n t h e d a t a s e t . From t h e m e a s u r e d c l a s s i f i c a t i o n f r e q u e n c i e s t h e s u r v i v a l and u n c e r t a i n t y i n s u r v i v a l were e s t i m a t e d . 3 . 3 . 3 P u r p o s e and p r o c e d u r e . The r e s u l t s o f e x p e r i m e n t s e t A i n d i c a t e d t h a t b e t a changed most r a p i d l y as a f u n c t i o n o f m i s o n i d a z o l e c o n c e n t r a t i o n i n t h e r a n g e o f 0 . 1 - 5 . 0 mM. I t i s n o t c l e a r w h e t h e r a l p h a a l s o c h a n g e d . E x p e r i m e n t s e t B was d e s i g n e d t o measure t h e e f f e c t o f m i s o n i d a z o l e i n t h i s c o n c e n t r a t i o n r a n g e more a c c u r a t e l y . F i v e v e s s e l s e a c h c o n t a i n i n g a d i f f e r e n t c o n c e n t r a t i o n o f m i s o n i d a z o l e (0 , 0 . 1 , 0 . 4 , 1 . 5 , and 5 . 0 mM) were i r r a d i a t e d . S u r v i v a l was a s s a y e d a t t e n d o s e s c o r r e s p o n d i n g a p p r o x i m a t e l y t o s u r v i v a l l e v e l s o f 1 . 0 , 0 . 8 5 , 0 . 7 0 . 6 , 0 . 5 , 0 . 2 , 0 . 1 , 0 . 0 3 , 0 . 0 1 , and 0 . 0 0 3 , t h e f i r s t f i v e o f w h i c h were a s s a y e d u s i n g t h e l o w dose a s s a y . The same p r o c e d u r e s were u s e d f o r e x p e r i m e n t s e t B as f o r e x p e r i m e n t s e t A w i t h t h e f o l l o w i n g m o d i f i c a t i o n s : 1 . I n o r d e r t o r e d u c e t h e u n c e r t a i n t y due t o v a r i a t i o n b e t w e e n e x p e r i m e n t s and t o o b t a i n a s t r a i g h t f o r w a r d e s t i m a t e o f t h i s v a r i a t i o n , s u r v i v a l was m e a s u r e d f o r a l l f i v e m i s o n i d a z o l e c o n c e n t r a t i o n s i n e a c h e x p e r i m e n t . I n a d d i t i o n , 10 mM m i s o n i d a z o l e s t o c k s o l u t i o n was u s e d t o p r e p a r e a l l s a m p l e s . 2 . One p e r s o n w o u l d i r r a d i a t e t h e s a m p l e s w h i l e a n o t h e r p e r s o n w o u l d p l a t e t h e i r r a d i a t e d s a m p l e s . I n t h i s way a l l f i v e i r r a d i a t i o n v e s s e l s w o u l d be i r r a d i a t e d and p l a t e d w i t h i n s e v e n h o u r s . The c e l l s f r o m t h e s p i n n e r c u l t u r e f l a s k were g r o w i n g e x p o n e n t i a l l y t h r o u g h o u t t h e d u r a t i o n o f t h e e x p e r i m e n t . A t y p i c a l t i m e c o u r s e f o r t h e e x p e r i m e n t i s shown i n f i g u r e 1 3 . 54 Figure 13: TIME COURSE OF EXPERIMENTS SET B. In order to reduce experimental e r r o r f o r experiment set B c e l l samples with f i v e d i f f e r e n t concentrations of misonidazole were i r r a d i a t e d each experiment and the time taken from the preparation of each sample of c e l l s to the p l a t i n g of the c e l l s was kept the same. To do t h i s i t was necessary to accurately time the experimental procedure as shown i n t h i s f i g u r e . The numbers represent the following concentrations of misonidazole: I - 0 mM; II - 0.1 mM; III - 0.4 mM; IV - 1.5 mM; V - 5.0 mM. 55 3 . C e l l s p l a t e d f o r t h e c o n v e n t i o n a l a s s a y were c e n t r i f u g e d f o r 6 m i n u t e s a t 600 r . p . m . p r i o r t o p l a t i n g t o remove t h e m i s o n i d a z o l e . C e l l s p l a t e d f o r t h e l o w d o s e a s s a y were n o t c e n t r i f u g e d s i n c e t h e s a m p l e s were d i l u t e d 2000 t o 5000 f o l d , r e d u c i n g t h e c o n c e n t r a t i o n o f m i s o n i d a z o l e b e l o w t h e t o x i c l e v e l . 4 . W i t h p r e v i o u s p r o c e d u r e s i t w o u l d be v e r y d i f f i c u l t t o measure f i v e s u r v i v a l c u r v e s f o r e a c h e x p e r i m e n t . The l i m i t i n g f a c t o r s were t h e p l a t i n g o f t h e m i c r o t e s t p l a t e s u s e d f o r t h e l o w dose a s s a y and t h e l o c a t i o n and c l a s s i f i c a t i o n o f t h e c e l l s p l a t e d i n t h e m i c r o t e s t p l a t e s . C e l l s f o r t h e 2 l o w d o s e a s s a y f o r e x p e r i m e n t s e t B were p l a t e d i n 25 cm a r e a f l a s k s t o s a v e t i m e i n p l a t i n g and c e l l l o c a t i o n . The a u t o m a t e d l o w dose a s s a y s y s t e m (ALDAS) was u s e d t o l o c a t e and c l a s s i f y c e l l s . W i t h ALDAS one c a n p l a t e , l o c a t e , c l a s s i f y , and compute t h e s u r v i v a l o f 100 t o 200 c e l l s p l a t e d i n a f l a s k i n 20 t o 30 m i n u t e s . P r e v i o u s l y t h i s a s s a y w o u l d t a k e a minimum o f 4 5 - 5 0 m i n u t e s and t h e r e s u l t s were q u a l i t a t i v e l y l e s s r e l i a b l e due t o o b s e r v e r s t r a i n i n l o c a t i n g more w i d e l y s p a c e d c e l l s u n d e r a l o w e r m a g n i f i c a t i o n t h r o u g h t h e m i c r o s c o p e . The d e t a i l s o f t h e m o d i f i e d l o w dose a s s a y p r o c e d u r e a r e p r e s e n t e d i n s e c t i o n 3 . 3 . 2 . 3 . 3 . 4 S u r v i v a l d a t a . The s u r v i v a l was e s t i m a t e d i n t h e same manner as f o r e x p e r i m e n t s e t A . T a b l e 2 s u m m a r i z e s t h e e x p e r i m e n t s e t B d a t a . The c h i - s q u a r e d t e s t was u s e d t o e s t i m a t e t h e most l i k e l y v a l u e s o f a l p h a and b e t a and t h e 70%, 95%, and 99 .9% c o n f i d e n c e i n t e r v a l s . D e t a i l s o f t h e d a t a a n a l y s i s and t h e r e s u l t s a r e p r e s e n t e d i n s e c t i o n 4 . 56 TABLE I I T a b l e I I : THE AVERAGED SURVIVAL DATA OF EXPERIMENT SET B . The s u r v i v a l as m e a s u r e d by t h e l o w dose a s s a y (LD) and t h e c o n v e n t i o n a l a s s a y (CV) was a v e r a g e d o v e r t h e t h r e e e x p e r i m e n t s o f e x p e r i m e n t s e t B . The r e s u l t s a r e shown b e l o w w i t h t h e e s t i m a t e d s t a n d a r d d e v i a t i o n . DOSE (Gy) SURVIVAL UNCERTAINTY DOSE (Gy) SURVIVAL UNCERTAINTY 0 mM M i s o n i d a z o l e : 0.1 mM M i s o n i d a z o l e : 0 L D / C V 1.00 0.00 0 L D / C V 0.98 0.02 161 LD 0.75 0.20 161 LD 0.92 0.25 236 LD 0.85 0.09 236 LD 0.69 0.04 310 LD 0.72 0.20 470 o L D 0.47 0.04 470 LD 0.68 0.08 631 ° LD 0.48 0.06 631 LD 0.56 0.06 814 L D / C V 0.36 0.06 814 L D / C V 0.37 0.06 1500 CV 0.11 0.06 1500 CV 0.18 0.13 1920 CV 0.032 0.011 1920 CV 0.089 0.057 2290 CV 0.0090 0.0070 2290 CV 0.033 0.009 2710 cv 0.0020 0.0006 2710 CV 0.0066 0.0010 3010 CV 0.0022 0.0004 0.4 mM M i s o n i d a z o l e : 1.5 mM M i s o n i d a z o l e : 0 L D / C V 1.01 0.02 0 L D / C V 1.00 0.00 206 LD 0.74 0.04 138 LD 0.87 0.08 287 LD 0.83 0.13 178 LD 0.78 0.03 413 LD 0.58 0.07 252 LD 0.78 0.03 550 LD 0.47 0.03 355 LD 0.57 0.03 688 L D / C V 0.31 0.06 458 LD 0.48 0.03 1210 cv 0.077 0.023 562 L D / C V 0.33 0.06 1520 cv 0.019 0.005 952 cv 0.088 0.012 1790 cv 0.0080 0.0036 1170 cv 0.040 0.015 2110 cv 0.0015 0.0011 1360 CV 0.017 0.008 2310 cv 0.00081 0.00024 1590 CV 0.0059 0.0031 1740 CV 0.00057 0.0003' 5.0 mM M i s o n i d a z o l e : 0 L D / C V 0.96 0.04 161 LD 0.80 0.04 229 LD 0.74 0.10 322 LD 0.69 0.06 414 LD 0.50 0.03 517 L D / C V 0.36 0.04 852 cv 0.067 0.012 1050 CV 0.025 0.006 1210 CV 0.0098 0.0029 1400 cv 0.0023 0.0010 1530 cv 0.00098 0.00057 57 RESULTS AND DISCUSSION 4 . 1 UNCERTAINTIES. 4 . 1 . 1 E x p e r i m e n t a l e r r o r , b i o l o g i c a l v a r i a t i o n , and e x p e r i m e n t e r  e r r o r . The u n c e r t a i n t i e s i n t h e s e e x p e r i m e n t s c a n be d i v i d e d i n t o t h r e e c l a s s e s , n a m e l y : 1 . Random e x p e r i m e n t a l e r r o r due t o v o l u m e t r i c m e a s u r e m e n t s , s t a t i s t i c a l f l u c t u a t i o n s i n c e l l c o u n t i n g , and u n c e r t a i n t y i n c e l l r e c o g n i t i o n . 2 . ' B i o l o g i c a l v a r i a t i o n ' b e t w e e n d o s e p o i n t s and e x p e r i m e n t a l d a y s w h i c h may o r may n o t be random. T h i s u n c e r t a i n t y i s p a r t l y due t o c h a n g e s i n t h e c e l l u l a r e n v i r o n m e n t (pH, c o n c e n t r a t i o n o f m e t a b o l i t e s , e t c . ) , c h a n g e s i n t h e n a t u r e o f t h e c e l l s ( g e n e t i c ) and t h e r e l a t i v e p r o p o r t i o n o f c e l l s i n e a c h s t a g e o f t h e c e l l c y c l e . Such e r r o r s a r e t h u s n o t i n c l u d e d i n t h e u n c e r t a i n t y e s t i m a t e s f o r i n d i v i d u a l e x p e r i m e n t s . 3 . E x p e r i m e n t e r e r r o r w h i c h l e a d s t o p o o r q u a l i t y d a t a . T h i s e r r o r r e s u l t s f r o m t h e i n a b i l i t y o f t h e e x p e r i m e n t e r t o r e p e a t t h e s t e p s o f t h e e x p e r i m e n t a l p r o c e d u r e w i t h minimum o f v a r i a t i o n b e t w e e n e x p e r i m e n t a l p o i n t s . T h i s e r r o r i s r e d u c e d by i m p r o v i n g t h e e x p e r i m e n t a l p r o c e d u r e and t h e s k i l l o f t h e e x p e r i m e n t e r . The r e s u l t s o f e x p e r i m e n t s e t A have g r e a t e r u n c e r t a i n t y t h a n t h o s e o f e x p e r i m e n t s e t B . T h i s i s i n p a r t due t o t h e r e d u c t i o n o f e x p e r i m e n t e r e r r o r t h r o u g h i m p r o v e d s k i l l and e x p e r i m e n t a l p r o c e d u r e . Those u n c e r t a i n t i e s r e s u l t i n g f r o m random e x p e r i m e n t a l e r r o r and b i o l o g i c a l v a r i a t i o n a r e i n c l u d e d i n t h e u n c e r t a i n t y e s t i m a t e s . An e x p e r i m e n t a l e r r o r i s e s t i m a t e d f o r e a c h s u r v i v a l measurement . B i o l o g i c a l v a r i a t i o n i s d e a l t w i t h a f t e r t h e r e s u l t s a r e o b t a i n e d . The r e s u l t s o f a p p r o x i m a t e l y h a l f o f t h e e x p e r i m e n t s p e r f o r m e d were d i s c a r d e d , m o s t l y due 58 t o b a c t e r i a l c o n t a m i n a t i o n b e f o r e i r r a d i a t i o n o f t h e c e l l s , d u r i n g i r r a d i a t i o n and p l a t i n g , o r d u r i n g i n c u b a t i o n a f t e r p l a t i n g . An u n c e r t a i n t y i s e s t i m a t e d f o r e a c h o f t h e f o l l o w i n g p a r a m e t e r s : t h e c o n c e n t r a t i o n o f m i s o n i d a z o l e , t h e dose o f r a d i a t i o n d e l i v e r e d t o t h e c e l l s , and t h e c e l l s u r v i v a l as m e a s u r e d by t h e l o w d o s e and t h e c o n v e n t i o n a l a s s a y . The u n c e r t a i n t y i n c e l l s u r v i v a l i s p r i m a r i l y due t o c o u n t i n g s t a t i s t i c s ( P o i s s o n ) and d i l u t i o n u n c e r t a i n t i e s so t h a t i t i s r e a s o n a b l e t o assume t h a t t h e e s t i m a t e o f c e l l s u r v i v a l , n o t t h e l o g a r i t h m o f c e l l s u r v i v a l , i s n o r m a l l y d i s t r i b u t e d a b o u t t h e mean. The s t a n d a r d d e v i a t i o n f r o m t h e e s t i m a t e o f t h e mean (63% c o n f i d e n c e i n t e r v a l i n t h e c a s e o f a n o r m a l d i s t r i b u t i o n ) i s e s t i m a t e d i n a l l c a s e s . 4 . 1 . 2 R a d i a t i o n d o s e r a t e and d r u g c o n c e n t r a t i o n u n c e r t a i n t i e s . The d o s e r a t e d e l i v e r e d by t h e P i c k e r X - r a y m a c h i n e was e s t i m a t e d u s i n g f e r r o u s s u l f a t e d o s i m e t r y t e c h n i q u e s ( F r i c k e and H a r t , 1966) t o be w i t h i n 5% o f t h e t r u e d o s e r a t e . The v a r i a t i o n i n t h e a c t u a l d o s e d e l i v e r e d i s c o n s i d e r e d t o be l e s s t h a n 1% o f t h e e s t i m a t e d d o s e . An u n c e r t a i n t y i n t h e dose r a t e w o u l d p r o d u c e t h e l a r g e s t u n c e r t a i n t y i n s u r v i v a l where t h e s l o p e o f t h e s u r v i v a l c u r v e i s t h e s t e e p e s t , t h a t i s , a t t h e l a r g e s t d o s e s and h i g h e s t m i s o n i d a z o l e c o n c e n t r a t i o n s where b e t a o f e q u a t i o n 3 i s l a r g e . From f i g u r e - 2 16 t h e maximum v a l u e o f b e t a i s e q u a l t o a b o u t 0 . 0 1 2 G r a y a t 5 . 0 mM m i s o n i d a z o l e and f r o m t a b l e 3 t h e maximum r a d i a t i o n dose i s 1 5 . 3 G r a y f o r 5 . 0 mM m i s o n i d a z o l e . A t t h i s d o s e i t c a n be shown t h a t a 1% v a r i a t i o n i n t h e d o s e r a t e w i l l n o t c o n t r i b u t e more t h a n 10% u n c e r t a i n t y t o t h e s u r v i v a l , and much l e s s a t l o w e r c o n c e n t r a t i o n s o f m i s o n i d a z o l e and a t l o w e r r a d i a t i o n d o s e s . U n c e r t a i n t y i n t h e dose r a t e i s c o n s i d e r e d t o be i n s i g n i f i c a n t and i s n o t i n c l u d e d i n t h e e r r o r e s t i m a t e s . M i s o n i d a z o l e s o l u t i o n s were p r e p a r e d w i t h g r e a t c a r e . The 59 c o n c e n t r a t i o n o f m i s o n i d a z o l e i n t h e medium s h o u l d n o t v a r y b y more t h a n 1% o f t h e q u o t e d v a l u e e x c e p t f o r a few e x p e r i m e n t s i n e x p e r i m e n t s e t A i n w h i c h 40 mM m i s o n i d a z o l e was p r e p a r e d . A l l o f t h e m i s o n i d a z o l e may n o t have d i s s o l v e d i n t h e s e s o l u t i o n s , r e s u l t i n g i n a l o w e r m i s o n i d a z o l e c o n c e n t r a t i o n t h a n q u o t e d . However , t h i s e x p e r i m e n t e r e r r o r was n o t r e p e a t e d i n e x p e r i m e n t s e t B w h i c h p r o v i d e s t h e s i g n i f i c a n t d a t a f o r t h i s s t u d y . U n c e r t a i n t y i n t h e c o n c e n t r a t i o n o f m i s o n i d a z o l e i s c o n s i d e r e d t o be i n s i g n i f i c a n t and i s n o t i n c l u d e d i n t h e e r r o r e s t i m a t e s . 4 . 1 . 3 S u r v i v a l a s s a y u n c e r t a i n t i e s . The s t a n d a r d d e v i a t i o n o f t h e c e l l s u r v i v a l depends on t h e a s s a y u s e d t o measure s u r v i v a l . The e r r o r o f t h e l o w dose a s s a y w i l l be d i s c u s s e d f i r s t : T h e r e a r e two s i g n i f i c a n t s o u r c e s o f e r r o r i n t h e l o w dose a s s a y . The c l a s s i f i c a t i o n ' p o s s i b l e e r r o r ' i s u s e d on c l a s s i f i c a t i o n day f o r c o l o n i e s t h a t e i t h e r c a n n o t be f o u n d a t t h e r e c o r d e d l o c a t i o n o r f o r c o l o n i e s t h a t l o o k l i k e d e b r i s ; i . e . , t h e y c o n t a i n no v i a b l e c e l l s . H a l f o f t h e ' p o s s i b l e e r r o r ' c o l o n i e s a r e c o u n t e d a s d e a d c o l o n i e s w i t h a s t a n d a r d d e v i a t i o n , s d ( e r r o r ) , e s t i m a t e d a s h a l f o f t h e ' p o s s i b l e e r r o r ' c o l o n i e s . Thus any e x p e r i m e n t a l v a r i a t i o n due t o l o c a t i o n e r r o r s w i l l be be a c c o u n t e d f o r i n t h e u n c e r t a i n t y e s t i m a t e s . The s e c o n d s i g n i f i c a n t s o u r c e o f e r r o r i s t h e s t a t i s t i c a l d i s t r i b u t i o n o f c e l l s d e s t i n e d t o s u r v i v e and c e l l s d e s t i n e d t o d i e w i t h i n t h e p l a t i n g v e s s e l . I f a c e l l h a s a p r o b a b i l i t y , p , o f d y i n g and a p r o b a b i l i t y , q , o f l i v i n g t h e n t h e d y i n g c e l l s a r e r a n d o m l y d i s t r i b u t e d i n a b i n o m i a l d i s t r i b u t i o n w i t h s t a n d a r d d e v i a t i o n , s d ( d e a t h ) = (Npq)^ E q u a t i o n 4 where N=number o f c e l l s . p = p r o b a b i l i t y t h a t a g i v e n c e l l i s a n o n - s u r v i v o r . q = l - p = p r o b a b i l i t y t h a t a g i v e n c e l l i s a s u r v i v o r . 60 From e q u a t i o n 2 t h e r e l a t i v e u n c e r t a i n t y i n t h e p r o d u c t o f t h e p l a t i n g e f f i c i e n c y and t h e s u r v i v a l i s : S d ( P S ) L D A _ sd (C ) 2 sd (N) 2 * PS C N a n d , s d ( C ) = s d ( d e a t h ) = (Npq) * sd(N) = s d ( e r r o r ) = E / 2 t h e r e f o r e , s d ( p s ) L D A = P S [ ( E / 2 N ) 2 + ( 1 / C - l / N ) ] * E q u a t i o n 5 w h e r e , N=C+D+E/2=number o f c e l l s p l a t e d . C=Nq=number o f s u r v i v o r s . D=number o f n o n - s u r v i v o r . E=number o f ' p o s s i b l e e r r o r ' c l a s s i f i c a t i o n s . Equation 5 was used to estimate the standard deviation of s u r v i v a l as measured by the low dose assay. There are three s i g n i f i c a n t sources of erro r i n the conventional assay. The estimate of the number of c e l l s p l ated i s subject to a counting uncertainty due to the Poisson d i s t r i b u t i o n of c e l l s i n the sample. For a Poisson d i s t r i b u t i o n , the standard deviation can be estimated by, sd(N) = Equation 6 N=number of c e l l s p l a t e d . The estimate of the number of surviving c e l l s , C , i s also subject to a counting uncertainty due to the Poisson d i s t r i b u t i o n of surviving c e l l s i n the sample. Also, as s u r v i v a l decreases there i s an increasing number of marginal colonies (colonies with close to 50 c e l l s ) . To take t h i s into account an estimate of the standard deviation i n the number of survivors due to uncertainty i n the colony c l a s s i f i c a t i o n was made for each colony '61 c o u n t i n w h i c h t h i s u n c e r t a i n t y was deemed s i g n i f i c a n t ( g r e a t e r t h a n a b o u t 10% o f t h e s u r v i v o r s ) . Thus t h e s t a n d a r d d e v i a t i o n o f t h e c o l o n y c o u n t c a n be e s t i m a t e d b y , s d ( C ) = (C + C 2 e 2 ) E q u a t i o n 7 w h e r e , e = e s t i m a t e d r e l a t i v e e r r o r due t o s u r v i v o r m i s c o u n t . From e q u a t i o n 1 , t h e r e l a t i v e u n c e r t a i n t y i n t h e p r o d u c t o f t h e p l a t i n g e f f i c i e n c y and t h e s u r v i v a l i s : S d ( P S ) C V L sd (C ) 2 sd (N) 2 PS C N t h e n , f r o m e q u a t i o n s 6 and 7 i t c a n be shown t h a t : sd(PS1 _ = P S ( 1 / N + l / C + e 2 ) ^ E q u a t i o n 8 w h e r e , N=number o f c e l l s p l a t e d . C=number o f s u r v i v o r s . e = e r r o r due t o s u r v i v o r m i s c o u n t . F o r t h e p u r p o s e o f t h e s e e x p e r i m e n t s , C i s l e s s t h a n o r a b o u t 150 , P = 0 . 6 , N=C /PS , and e becomes s i g n i f i c a n t a t s u r v i v a l l e v e l s o f l e s s t h a n 10%. T h e r e f o r e s d ( P S ) / P S i s a t l e a s t 0 . 1 0 f o r l a r g e S ( a p p r o a c h i n g u n i t y ) , and f o r s m a l l S , e becomes l a r g e and 1 /N becomes i n s i g n i f i c a n t compared t o 1 / C , s o t h a t e q u a t i o n 8 s i m p l i f i e s t o : s d ( P S ) I _ _ C V L = M A X [ 0 . 1 0 , ( l / C + e ) 2 ] E q u a t i o n 9 PS E q u a t i o n 9 was u s e d t o e s t i m a t e d t h e u n c e r t a i n t i e s o f s u r v i v a l as measu red w i t h t h e c o n v e n t i o n a l a s s a y . N o t e t h a t i n o r d e r t o e s t i m a t e t h e s t a n d a r d d e v i a t i o n i n t h e n o r m a l i z e d s u r v i v a l , i t i s a l s o n e c e s s a r y t o t a k e i n t o 62 a c c o u n t t h e u n c e r t a i n t y i n t h e n o r m a l i z a t i o n f a c t o r P ( p l a t i n g e f f i c i e n c y ) , w h i c h i s o b t a i n e d f r o m e q u a t i o n 9 when t h e s u r v i v a l S = l . The f o l l o w i n g i s a c o m p a r a t i v e u n c e r t a i n t y a n a l y s i s o f t h e s e two a s s a y s : I f t h e u n c e r t a i n t y i n t h e p l a t i n g e f f i c i e n c y , P , i s t a k e n i n t o a c c o u n t , ( the u n c e r t a i n t y i n P i s e s t i m a t e d f r o m e q u a t i o n 5 f o r z e r o r a d i a t i o n d o s e , S = l ) , and i f t h e number o f ' p o s s i b l e e r r o r ' c l a s s i f i c a t i o n s i n t h e l o w d o s e a s s a y , E , i s k e p t s m a l l , t h e n t h e r e l a t i v e u n c e r t a i n t y i n t h e l o w d o s e a s s a y f r o m e q u a t i o n 5 i s : S d ( S ) L D A _ s d ( P S ) 2 s d ( P ) » * S PS P = [__1 1 _ 1 _ 1 ] * N PS N N P ~ N LDA LDA LDA LDA t h e r e f o r e , s d L D A _ JL r^Ltl. + n 9 1 S ~ ( N L D A E q u a t i o n 10 w h e r e , C=NPS s i n c e , N L D A = n u m b e r o f c e l l s p l a t e d , i s ( a p p r o x i m a t e l y ) i n d e p e n d e n t o f t h e s u r v i v a l l e v e l f o r t h e l o w l o w dose a s s a y . S i m i l a r i l y i f t h e u n c e r t a i n t y i n t h e c o n v e n t i o n a l a s s a y p l a t i n g e f f i c i e n c y , Q, i s t a k e n i n t o a c c o u n t ( u s i n g e q u a t i o n 9 ) , i f t h e e r r o r due t o s u r v i v o r m i s c o u n t , e , i n t h e c o n v e n t i o n a l a s s a y i s k e p t s m a l l , and i f t h e number o f s u r v i v i n g c o l o n i e s p e r p e t r i s d i s h i s ( a p p r o x i m a t e l y ) i n d e p e n d e n t o f s u r v i v a l , t h e n t h e r e l a t i v e u n c e r t a i n t y i n t h e c o n v e n t i o n a l a s s a y f r o m e q u a t i o n 8 i s a p p r o x i m a t e l y : S d ( S ) C V L 1 2 1 L V L = (jL-r± + s + 11 ) * S o + B + ^-J E q u a t i o n 11 w h e r e , Q = c o n v e n t i o n a l a s s a y p l a t i n g e f f i c i e n c y N =N S=number o f c e l l s p l a t e d a t z e r o o . C V L ^ . _ , ^ ^ i r r a d i a t i o n dose i f C i s i n d e p e n d e n t o f s u r v i v a l . 63 The two s u r v i v a l a s s a y s c a n be compared by e s t i m a t i n g t h e r a t i o o f t h e number o f c e l l s w h i c h must b e p l a t e d i n o r d e r t o a c h i e v e t h e same a c c u r a c y . T h i s i s done by e q u a t i n g sd (S ) f r o m e q u a t i o n 10 w i t h s d ( S ) f r o m LDA C VL e q u a t i o n 11 w h i c h g i v e s t h e f o l l o w i n g r e s u l t : N C V L _ [2 /Q + S + 1] E q u a t i o n 12 N L D A = [ ( 1 + S ) / P " 2 S ] Now, f o r t h e s e e x p e r i m e n t s , Q i s a b o u t 0 . 6 0 and P i s a b o u t 0 . 9 0 so t h a t a t a s u r v i v a l l e v e l o f S=0.5 a p p r o x i m a t e l y s e v e n t i m e s a s many c e l l s a r e r e q u i r e d by t h e c o n v e n t i o n a l a s s a y t o y i e l d as a c c u r a t e a r e s u l t as t h a t a t t a i n a b l e w i t h t h e l o w dose a s s a y , and f o r S=0.9 a p p r o x i m a t e l y f i f t e e n t i m e s as many c e l l s a r e r e q u i r e d . E v e n i f t h e c o n v e n t i o n a l a s s a y p l a t i n g e f f i c i e n c y , Q, was i m p r o v e d t h r o u g h more a c c u r a t e means o f e s t i m a t i n g t h e d e n s i t y o f c e l l s i n s u s p e n s i o n s o t h a t Q=P, a p p r o x i m a t e l y t e n t i m e s a s many c e l l s a r e r e q u i r e d by t h e c o n v e n t i o n a l a s s a y t o y i e l d as a c c u r a t e a r e s u l t a s t h e l o w d o s e a s s a y a t a s u r v i v a l l e v e l o f S = 0 . 8 . 4 . 1 . 4 The t o t a l u n c e r t a i n t y i n s u r v i v a l . S u r v i v a l i s measu red by e i t h e r t h e l o w dose o r t h e c o n v e n t i o n a l a s s a y . The random ' e x p e r i m e n t a l e r r o r ' i s e s t i m a t e d f r o m e q u a t i o n 5 f o r t h e l o w dose a s s a y o r e q u a t i o n 9 f o r t h e c o n v e n t i o n a l a s s a y f o r e a c h s u r v i v a l p o i n t . The a d d i t i o n a l e r r o r due t o ' b i o l o g i c a l v a r i a t i o n ' c a n o n l y be e s t i m a t e d a f t e r a l l o f t h e d a t a i s c o l l e c t e d . The t o t a l u n c e r t a i n t y i n s u r v i v a l i s due t o t h e ' e x p e r i m e n t a l e r r o r ' and t h e ' b i o l o g i c a l v a r i a t i o n ' . The most s t r a i g h t f o r w a r d way t o e s t i m a t e t h e t o t a l u n c e r t a i n t y i n s u r v i v a l i s t o measure t h e s u r v i v a l a t e a c h dose f o r e a c h m i s o n i d a z o l e c o n c e n t r a t i o n s e v e r a l t i m e s . M o s t o f t h e s u r v i v a l d a t a o f e x p e r i m e n t s e t B i s a c o m b i n a t i o n o f two o r more d a t a p o i n t s . I f t h e s e d a t a p o i n t s a r e n o r m a l l y 64 d i s t r i b u t e d a b o u t t h e mean s u r v i v a l t h e s t a n d a r d d e v i a t i o n i n s u r v i v a l c a n be e s t i m a t e d as f o l l o w s ( B e v i n g t o n , 1 9 6 9 ) : _ ' (q _ q } 2 i s d ( S . ) = k=l,Nv k I * 1 N ( N - l ) E q u a t i o n 13 S ^ = S u r v i v a l , k t h d a t a p o i n t . S . = A v e r a g e s u r v i v a l a t a g i v e n X - r a y dose m i s o n i d a z o l e c o n c e n t r a t i o n . N=number o f d a t a p o i n t s . However , e a c h d a t a p o i n t h a s an a s s o c i a t e d e x p e r i m e n t a l e r r o r , s d ( S ) . K. I f t h e b i o l o g i c a l v a r i a t i o n i s s m a l l compared t o t h e e x p e r i m e n t a l e r r o r i t c a n be n e g l e c t e d and t h e s t a n d a r d d e v i a t i o n i n s u r v i v a l c a n be e s t i m a t e d a s f o l l o w s ( B e v i n g t o n , 1 9 6 9 ) : l / s d ( S ± ) = [ l / s d ( S k ) ) 2 E q u a t i o n 14 w h e r e , s d ( S ^ ) = e x p e r i m e n t a l e r r o r o f k t h d a t a p o i n t . s d ( S . ) = u n c e r t a i n t y i n a v e r a g e d s u r v i v a l a t i t h d o s e p o i n t . S i n c e t h e s t a n d a r d d e v i a t i o n i n s u r v i v a l c a n n o t be l e s s t h a n s d ( S ) f r o m e q u a t i o n 14 t h e u n c e r t a i n t y i n s u r v i v a l f o r e a c h X - r a y dose p o i n t o f e x p e r i m e n t s e t B w i t h two o r more measurements o f s u r v i v a l i s e s t i m a t e d a s t h e maximum v a l u e o f s d ( S ^ ) f r o m e q u a t i o n s 13 and 14 . I n g e n e r a l , f o r t h e e x p e r i m e n t s e t B d a t a , t h e t o t a l e s t i m a t e d u n c e r t a i n t y f r o m e q u a t i o n 13 was g r e a t e r t h a n t h a t f r o m e q u a t i o n 14 . T h i s was e x p e c t e d and i s due t o t h e s i g n i f i c a n t v a r i a t i o n b e t w e e n s u r v i v a l measurements made on d i f f e r e n t d a y s , t h e ' b i o l o g i c a l v a r i a t i o n ' . T h i s r e s u l t a l s o p o i n t s o u t t h a t i f an e x p e r i m e n t i s r e p e a t e d a t l e a s t t h r e e t i m e s , t h e u n c e r t a i n t y i n t h e a v e r a g e s u r v i v a l i s b e s t e s t i m a t e d f r o m e q u a t i o n 1 3 . T h e r e f o r e t h e t e d i o u s 65 u n c e r t a i n t y c a l c u l a t i o n s u s e d t o e s t i m a t e e x p e r i m e n t a l e r r o r need n o t be c a r r i e d o u t . I f o n l y one d a t a p o i n t i s o b t a i n e d f o r a p a r t i c u l a r r a d i a t i o n d o s e and m i s o n i d a z o l e c o n c e n t r a t i o n a n o t h e r means o f e s t i m a t i n g t h e t o t a l u n c e r t a i n t y ( w h i c h i n c l u d e s ' e x p e r i m e n t a l e r r o r ' and ' b i o l o g i c a l v a r i a t i o n ' ) i s r e q u i r e d . To do t h i s i t was assumed t h a t e q u a t i o n 3 was a s u i t a b l e f i t t o t h e s u r v i v a l d a t a . The most l i k e l y v a l u e s f o r a l p h a and 2 b e t a were e s t i m a t e d by m i n i m i z i n g t h e v a r i a b l e X o f e q u a t i o n 15 ( a f t e r B e v i n g t o n , 1 9 6 9 ) . 2 S - S ( a , b ) 2 1 ' 1 1 s d ( S i ) J E q u a t i o n 15 and S i s ^ a , b ^ = t h e r e s i d u a l s . E q u a t i o n 16 s d ( S . ) l r-i v-v , A - a D - b D 2 w h e r e , S ( a , b ) = 1 0 S . = s u r v i v a l , i t h d a t a p o i n t . so l (S . ) = e s t i m a t e d s t a n d a r d d e v i a t i o n . N=number o f d a t a p o i n t s . D = r a d i a t i o n d o s e . The r e s i d u a l s ( e q u a t i o n 16) were t h e n c a l c u l a t e d f o r e a c h s u r v i v a l , S ^ , and p l o t t e d a g a i n s t s u r v i v a l . N e i t h e r t h e mean n o r t h e s t a n d a r d d e v i a t i o n i n t h e r e s i d u a l s showed a s i g n i f i c a n t dependence on s u r v i v a l ; h o w e v e r , t h e s p r e a d i n t h e d a t a p o i n t s i n d i c a t e d t h a t t h e t o t a l u n c e r t a i n t y i n t h e s u r v i v a l was two t o t h r e e t i m e s t h e e x p e r i m e n t a l u n c e r t a i n t y . I t was t h e n assumed t h a t t h e r e s i d u a l s were n o r m a l l y d i s t r i b u t e d and t h e means and s t a n d a r d d e v i a t i o n s o f t h e r e s i d u a l s were c a l c u l a t e d . The d a t a o f e x p e r i m e n t s e t A gave a mean o f 0.1±2.3 and t h e d a t a o f e x p e r i m e n t s e t B gave a mean o f 0.5±2.5. Thus i t i s r e a s o n a b l e t o e s t i m a t e t h e t o t a l s t a n d a r d d e v i a t i o n o f e a c h d a t a p o i n t t o be a b o u t 2 . 5 t i m e s t h e e s t i m a t e d 66 e x p e r i m e n t a l u n c e r t a i n t y as f o l l o w s : s d ( S ^ ) = 2 . 5 x ( e s t i m a t e d e x p e r i m e n t a l u n c e r t a i n t y ) E q n . 17 F o r e x p e r i m e n t s e t A e q u a t i o n 17 was u s e d t o e s t i m a t e t h e t o t a l s t a n d a r d d e v i a t i o n o f e a c h d a t a p o i n t f o r d a t a s e t s w i t h more t h a n one e x p e r i m e n t s i n c e t h e v a r i a t i o n b e t w e e n e x p e r i m e n t s , o r ' b i o l o g i c a l ' v a r i a t i o n h a d t o be t a k e n i n t o a c c o u n t . F o r d a t a s e t s w i t h o n l y one e x p e r i m e n t t h e t o t a l s t a n d a r d d e v i a t i o n was e s t i m a t e d as t h e e s t i m a t e d e x p e r i m e n t a l e r r o r . I n t h e s e c a s e s t h e ' b i o l o g i c a l v a r i a t i o n ' was c o n s i d e r e d t o be i n s i g n i f i c a n t . F o r e x p e r i m e n t s e t B e q u a t i o n 17 was u s e d as t h e e s t i m a t e o f t h e t o t a l s t a n d a r d d e v i a t i o n i f t h e r e was o n l y one d a t a p o i n t , o t h e r w i s e t h e maximum v a l u e o f e q u a t i o n s 13 and 14 was u s e d . 4 . 2 RESULTS. 4 . 2 . 1 Method o f p a r a m e t e r e s t i m a t i o n . The s u r v i v a l d a t a o f e x p e r i m e n t s e t B a r e t a b u l a t e d i n t a b l e 2 . F o r e x p e r i m e n t s e t B t h e s u r v i v a l a t e a c h r a d i a t i o n dose and m i s o n i d a z o l e c o n c e n t r a t i o n i s t h e a v e r a g e measu red s u r v i v a l . The most l i k e l y v a l u e s o f a l p h a and b e t a o f e q u a t i o n 3 were e s t i m a t e d by m i n i m i z i n g t h e v a r i a b l e X 2 o f e q u a t i o n 15 where s d ( S ^ ) i s t h e t o t a l s t a n d a r d d e v i a t i o n o f t h e s u r v i v a l . S i n c e t h e s u r v i v a l a t z e r o r a d i a t i o n d o s e was g e n e r a l l y m e a s u r e d s e v e r a l t i m e s f o r e a c h m i s o n i d a z o l e c o n c e n t r a t i o n i t was c o n s t r a i n e d t o u n i t y . T h i s w i l l n o t s i g n i f i c a n t l y a f f e c t t h e e s t i m a t e s o f a l p h a and b e t a b u t g r e a t l y s i m p l i f i e s t h e d a t a a n a l y s i s . I f t h e s u r v i v a l v a l u e s , S ^ , a r e n o r m a l l y d i s t r i b u t e d w i t h s t a n d a r d d e v i a t i o n s d ( S ^ ) t h e n t h e v a r i a b l e X 2 h a s a c h i - s q u a r e d 67 d i s t r i b u t i o n f o r w h i c h t h e number o f d e g r e e s o f f r e e d o m i s t h e number o f n o n - z e r o d o s e p o i n t s i n t h e d a t a s e t m i n u s t h r e e , ( s i n c e two p a r a m e t e r s w e r e e s t i m a t e d f o r e a c h d a t a s e t ) , and t h e most l i k e l y v a l u e s o f t h e p a r a m e t e r s a l p h a and b e t a a r e t h o s e w h i c h r e s u l t i n t h e minimum v a l u e o f X 2 . ( B e v i n g t o n , 1 9 7 6 ) . The minimum v a l u e o f t h e f u n c t i o n X 2 ( a , b ) was e s t i m a t e d as f o l l o w s : A c o m p u t e r p r o g r a m was w r i t t e n t o c a l c u l a t e t h e v a l u e s o f X 2 f o r a s e t o f s u r v i v a l d a t a on an a r r a y o f a l p h a and b e t a v a l u e s . The s i z e o f t h e a r r a y , NxM, and t h e r a n g e o f a l p h a and b e t a was d e f i n e d by t h e u s e r . The p r o g r a m w o u l d d i v i d e t h e r a n g e o f a l p h a i n t o N v a l u e s s e p a r a t e d by e q u a l i n c r e m e n t s , t h e r a n g e o f b e t a i n t o M v a l u e s s e p a r a t e d by e q u a l i n c r e m e n t s and c a l c u l a t e X 2 a t e a c h a l p h a and b e t a v a l u e i n t h e a r r a y f r o m t h e s u r v i v a l d a t a u s i n g e q u a t i o n 1 5 . A s e c o n d p r o g r a m was w r i t t e n t o c o n t o u r t h e a r r a y o f X 2 v a l u e s on a g r a p h o f a l p h a v e r s u s b e t a . The p r o c e d u r e u s e d was t o f i r s t a p p r o x i m a t e l y l o c a t e t h e minimum o f X 2 u s i n g an a r r a y o f 41 a l p h a v a l u e s s e p a r a t e d by 0 . 0 0 5 Gy ^ and 61 b e t a v a l u e s s e p a r a t e d b y - 2 0 . 0 0 0 5 Gy . T h i s g e n e r a l l y a l l o w e d f o r s u f f i c i e n t r a n g e i n t h e a l p h a and b e t a p a r a m e t e r s i n o r d e r t o c o n t o u r t h e 70%, 95%, and 99.9% c o n f i d e n c e i n t e r v a l s on t h e e s t i m a t e s o f t h e s e p a r a m e t e r s . N e x t a f i n e r a r r a y o f 21 a l p h a v a l u e s s e p a r a t e d by 0 . 0 0 1 Gy ^ and 31 b e t a v a l u e s s e p a r a t e d b y - 2 0 . 0 0 0 1 Gy was u s e d t o l o c a t e d t h e minimum X 2 w i t h a r e s o l u t i o n o f - 1 - 2 ±0.001 Gy i n a l p h a and ±0.0001 Gy i n b e t a . G r e a t e r r e s o l u t i o n was c o n s i d e r e d u n e c e s s a r y b e c a u s e t h e o b s e r v e d u n c e r t a i n t y i n t h e e s t i m a t e s o f t h e s e p a r a m e t e r s was much l a r g e r t h a n t h e u n c e r t a i n t y i n t r o d u c e d by t h e g r i d . The c o s t o f d a t a a n a l y s i s was e x p e n s i v e (about $ 5 0 . 0 0 t o d e v e l o p t h e p r o g r a m s and $ 1 5 0 . 0 0 t o c a l c u l a t e t h e r e s u l t s ) . The m a j o r c o s t was due t o c a l c u l a t i n g t h e a r r a y o f X 2 v a l u e s . C o s t c o u l d be r e d u c e d by u s i n g a more 68 e f f i c i e n t f u n c t i o n m i n i m i z a t i o n r o u t i n e . I u n s u c c e s s f u l l y t r i e d one s u c h r o u t i n e w r i t t e n f o r t h e U n i v e r s i t y o f V i c t o r i a IBM 370 c o m p u t e r . The d i f f i c u l t y l a y i n t h e l a r g e v a l u e s w h i c h X 2 w o u l d t a k e on i n r e g i o n s o f a l p h a and b e t a n e a r t h e minimum. S i n c e t h e f u n c t i o n X 2 i s an e l l i p s e w i t h i t s m a j o r a x i s r u n n i n g d i a g o n a l w i t h r e s p e c t t o t h e a l p h a and b e t a a x e s l a r g e v a l u e s o f X 2 o c c u r v e r y q u i c k l y i n t h e d i r e c t i o n o f t h e m i n o r a x i s o f t h e e l l i p s e . I t i s v e r y d i f f i c u l t t o c o n s t r a i n a l p h a and b e t a t o a r e g i o n where X 2 i s n o t l a r g e s i n c e t h i s r e g i o n i s e l l i p t i c a l . The p r o g r a m w o u l d t h e n t e r m i n a t e due t o u n d e r f l o w e r r o r s . C o n s e q u e n t l y t h i s more a n a l y u t i c a l method o f f u n c t i o n m i n i m i z a t i o n was a b a n d o n e d . The u n d e r f l o w e r r o r was a r e s u l t o f t h e p a r a m e t e r s b e i n g i n t h e e x p o n e n t o f t h e s u r v i v a l e q u a t i o n . I f t h e l o g a r i t h m o f t h e s u r v i v a l was u s e d i n s t e a d , t h e c h i - s q u a r e d f u n c t i o n X 2 L Q G ( e q u a t i o n 18) w o u l d n o t i n c r e a s e so r a p i d l y and w o u l d be s u i t a b l e f o r f u n c t i o n m i n i m i z a t i o n a l g o r i t h m s . more x 2 = l o g ( S . ) - a D - b D 2 2 L 0 G " i = 1' N [ slhsT] ] E q u a t i o n 18 The p r o b l e m w i t h e q u a t i o n 18 i s t h a t t h e u n c e r t a i n t i e s i n t h e l o g a r i t h m o f s u r v i v a l a r e n o t n o r m a l l y d i s t r i b u t e d . T h i s i s p a r t i c u l a r i l y n o t i c e a b l e f o r t h e i m p o r t a n t l o w d o s e , h i g h s u r v i v a l d a t a . C o n s e q u e n t l y X 2 L 0 G i s n o t a c h i - s q u a r e d d i s t r i b u t i o n . I f e q u a t i o n 18 was u s e d t h e v a l i d i t y o f t h e q u a l i t y o f f i t ( P ( X 2 , v ) ) and c o n f i d e n c e i n t e r v a l s r e s u l t s w o u l d have b e e n j e o p a r d i z e d . However , t h i s may have l e d t o a q u i c k e r , l e s s e x p e n s i v e method o f e s t i m a t i n g t h e b e s t f i t v a l u e s o f a l p h a and b e t a and t h e d i s t r i b u t i o n o f t h e l o g a r i t h m o f s u r v i v a l may have b e e n s u f f i c i e n t l y n o r m a l t o w a r r a n t s u c h an a n a l y s i s . Many r e s e a r c h e r s use e q u a t i o n 18 f o r t h e i r d a t a a n a l y s i s ( e . g . , C h a d w i c k and L e e n h o u t s , 1 9 8 1 ; K e l l e r e r and B r e n o t , 69 1974) . However , i t s u s e h a s n o t b e e n j u s t i f i e d i n t h e c a s e o f d a t a w i t h h i g h s u r v i v a l l e v e l s . T h i s h a s b e e n l e f t f o r f u t u r e c o n s i d e r a t i o n . The l e a s t s q u a r e s method o f p a r a m e t e r e s t i m a t i o n i s a much e a s i e r a p p r o a c h and some may wonder why t h i s method was n o t u s e d . The l e a s t s q u a r e s method assumes t h a t e a c h d a t a p o i n t has t h e same s t a n d a r d d e v i a t i o n and t h e r e f o r e d e f i n e s t h e b e s t f i t v a l u e s o f a l p h a and b e t a as t h o s e w h i c h m i n i m i z e t h e f u n c t i o n X 2 o f e q u a t i o n 18 w i t h t h e L O G u n c e r t a i n t y t e r m s e t t o a c o n s t a n t . Then t h e v a l u e o f t h i s f u n c t i o n i s an e s t i m a t e o f t h e s t a n d a r d d e v i a t i o n o f t h e p a r a m e t e r s . ( B e v i n g t o n , 1 9 7 6 ) . I n p r a c t i c e t h i s a p p r o a c h f a v o u r s t h e l o w e r q u a l i t y h i g h d o s e d a t a o v e r t h e more a c c u r a t e l o w dose d a t a . F o r t h e d a t a o f e x p e r i m e n t s e t B t h e l e a s t s q u a r e s f i t gave r e s u l t s w i t h i n t h e 70% c o n f i d e n c e l i m i t s o f t h e p a r a m e t e r s as e s t i m a t e d by t h e c h i - s q u a r e d m i n i m i z a t i o n m e t h o d , b u t o n l y i f t h e s u r v i v a l a t z e r o dose was c o n s t r a i n e d t o u n i t y . The l e a s t s q u a r e s f i t f a i l e d m i s e r a b l y i n i t s e s t i m a t e s o f t h e s t a n d a r d d e v i a t i o n o f t h e p a r a m e t e r s , g i v i n g u n c e r t a i n t y l i m i t s w h i c h were a t l e a s t an o r d e r o f m a g n i t u d e s m a l l e r t h a n t h e o b s e r v e d v a r i a t i o n i n t h e s e p a r a m e t e r s . C o n s e q u e n t l y t h i s a p p r o a c h s h o u l d be r e s e r v e d f o r p a r a m e t e r e s t i m a t i o n o n l y . O t h e r w i s e an a p p r o a c h w h i c h a l l o w s v a r i a t i o n i n t h e s u r v i v a l u n c e r t a i n t y w i t h d o s e i s p r e f e r a b l e . The minimum v a l u e s o f X 2 , t h e v a l u e s o f t h e p a r a m e t e r s a l p h a and b e t a w h i c h y i e l d t h i s minimum v a l u e , and t h e p r o b a b i l i t y o f o b t a i n i n g t h i s v a l u e i f t h e f u n c t i o n i s a good f i t ( f rom t a b l e C - 4 o f B e v i n g t o n , 1976) a r e shown i n t a b l e 3 . 4 . 2 . 2 Method o f c o n f i d e n c e e s t i m a t i o n . I f e q u a t i o n 3 i s t h e c o r r e c t m a t h e m a t i c a l m o d e l (o r a good a p p r o x i m a t i o n ) f o r t h e s u r v i v a l o f CHO c e l l s u n d e r t h e c o n d i t i o n s o f t h i s e x p e r i m e n t and i f t h e s u r v i v a l d a t a , S ^ , a r e n o r m a l l y d i s t r i b u t e d w i t h t o t a l s t a n d a r d d e v i a t i o n s d ( S j t h e n X 2 o f 70 TABLE III Table I I I : CHI-SQUARE FIT RESULTS. The value of the chi-squared function, equation 15, was calculated f o r a g r i d of alpha and beta values f o r the s u r v i v a l data obtained f o r each concentration of misonidazole. The function was contoured at the i n t e r v a l s shown i n the table, corresponding to confidence i n t e r v a l s i n the estimates of the alpha and beta parameters of 70%, 95% and 99.9%. These contours are shown i n figures 14 through 17. The values of alpha and beta which gave a minimum value of the chi-squared function are shown along with the minimum value. The p r o b a b i l i t y of obtaining a value of chi-squared greater than or equal to t h i s value ( i f equation 3 i s the correct f i t to the data) was determined from table C-4 of Bevington (1976). This p r o b a b i l i t y (PBY) i s shown i n the l a s t column of the tab l e . MISONIDAZOLE X CONTOUR ALPHA BETA MINIMUM PBY CONCENTRATION VALUES (/Gy) (/Gy2) VALUE OF (mM) +0.001 ±0.0001 X 2 EXPERIMENT SET A RESULTS: 0.0 28.3 37.8 52.5 0.020 0.0026 25.8 0.31 0.05 9.52 15.5 26. 2 0.026 0.0022 8.5 0.21 0.10 19.5 27.5 40.8 0.053 0.0022 5.5 0.99 0.25 18.4 26.2 39.2 0.035 0.0030 3.0 0.99 0.50 19.5 27.5 40.8 0.052 0.0059 16.3 0.36 1.0 25.9 35.2 49.8 0.018 0.0072 35.8 0.02 3.0 28.3 37.8 52.5 0.022 0.0170 25.9 0.14 5.0 27.2 36.5 51.2 0.059 0.0092 12.8 0.93 10.0 9.52 15.5 26.2 0.082 0.0129 3.75 0.71 40.0 9.52 15.5 26.2 0.055 0.0074 9.37 0.16 EXPERIMENT SET B RESULTS: 0.0 11.8 18.3 29.6 0.029 0.0019 0.55 0.82 0.1 10.7 16.9 27.9 0.052 0.0018 1.39 0.21 0.4 10.7 16.9 27.9 0.042 0.0043 0.79 0.60 1.5 10.7 16.9 27.9 0.039 0.0078 0.73 0.65 5.0 11.8 18.3 29.6 0.027 0.0118 0.62 0.76 71 e q u a t i o n 15 h a s a c h i - s q u a r e d d i s t r i b u t i o n and t h e p r o b a b i l i t y o f o b t a i n i n g a s e t o f d a t a s u c h t h a t X 2 e x c e e d s a s p e c i f i e d v a l u e c a n b e q u a n t i t a t i v e l y d e t e r m i n e d . ( B e v i n g t o n , 1 9 7 6 ) . The number o f d e g r e e s o f f r e e d o m i s now t h e number o f n o n - z e r o r a d i a t i o n dose p o i n t s m inus o n e . The p a r a m e t e r s a l p h a and b e t a a r e n o t a l l o w e d t o v a r y b u t a r e f i x e d and t h e r e f o r e do n o t r e d u c e t h e number o f d e g r e e s o f f r e e d o m . T a b l e C - 4 o f B e v i n g t o n (1976) was u s e d t o d e t e r m i n e t h e v a l u e s o f X 2 f o r a 70%, 95%, and 99 .9% c o n f i d e n c e i n t e r v a l . F o r e x a m p l e , f o r a d a t a s e t w i t h e l e v e n d a t a p o i n t s ( t e n d e g r e e s o f f r e e d o m ) , X 2 i s g r e a t e r t h a n 1 1 . 8 w i t h p r o b a b i l i t y 0 . 3 , X 2 i s g r e a t e r t h a n 1 8 . 3 w i t h p r o b a b i l i t y 0 . 0 5 , and X 2 i s g r e a t e r t h a n 2 9 . 6 w i t h p r o b a b i l i t y 0 . 0 1 g i v i n g t h e 70%, 95%, and 99 .9% c o n f i d e n c e i n t e r v a l s r e s p e c t i v e l y . The f u n c t i o n X 2 ( a , b ) was c a l c u l a t e d on a g r i d o f 41 a l p h a and 61 b e t a v a l u e s and c o n t o u r e d a t t h e 70%, 95%, and 99.9% c o n f i d e n c e l i m i t s . (The r a n g e o f t h e a l p h a and b e t a p a r a m e t e r s depended on t h e l o c a t i o n o f t h e minimum o f t h e X 2 f u n c t i o n f o r t h e d a t a s e t b e i n g c o n t o u r e d . ) The r e s u l t was a s e t o f t h r e e c o n c e n t r i c c l o s e d c u r v e s ( e r r o r e l l i p s e s ) f o r e a c h m i s o n i d a z o l e c o n c e n t r a t i o n . The c o n f i d e n c e e s t i m a t e s a r e p l o t t e d i n f i g u r e 14 (70% c o n f i d e n c e l i m i t s o n l y ) f o r e x p e r i m e n t s e t A and f i g u r e s 16 (70% c o n f i d e n c e l i m i t s ) , 17 (95% c o n f i d e n c e l i m i t s ) , and 18 (99.9% c o n f i d e n c e l i m i t s ) f o r e x p e r i m e n t s e t B. The v a l u e o f X 2 f o r t h e c o n t o u r s drawn and t h e r a n g e i n t h e p a r a m e t e r s a l p h a and b e t a a r e shown i n t a b l e 3 . The r e s o l u t i o n o f t h e g r i d f o r e a c h m i s o n i d a z o l e c o n c e n t r a t i o n i s ±0.005 Gy ^ - 2 i n a l p h a and ±0.0005 Gy i n b e t a . 4 . 2 . 3 R e s u l t s o f p a r a m e t e r and c o n f i d e n c e e s t i m a t e s . The r e s i d u a l s o f t h e d a t a s e t s f o r 0 mM and 5 mM m i s o n i d a z o l e c o n c e n t r a t i o n s f r o m b o t h e x p e r i m e n t s e t s w e r e o b t a i n e d f r o m e q u a t i o n 16 w i t h s d ( S j e q u a l t o t h e e s t i m a t e d e x p e r i m e n t a l s t a n d a r d d e v i a t i o n . Q u a l i t a t i v e l y t h e r e s i d u a l s 72 t e n d e d t o be n o r m a l l y d i s t r i b u t e d . The mean o f t h e d i s t r i b u t i o n was 0.1±2.3 s t a n d a r d d e v i a t i o n f o r e x p e r i m e n t s e t A and 0.5±2.5 s t a n d a r d d e v i a t i o n f o r e x p e r i m e n t s e t B. Thus t h e t o t a l e x p e r i m e n t a l u n c e r t a i n t y was on t h e a v e r a g e a p p r o x i m a t e l y 2 . 5 t i m e s t h e e s t i m a t e d e x p e r i m e n t a l u n c e r t a i n t y . T h e r e i s a s l i g h t t e n d e n c y f o r d a t a p o i n t s t o have s u r v i v a l v a l u e s g r e a t e r t h a n t h o s e p r e d i c t e d by e q u a t i o n 3 (S^ g r e a t e r t h a n S ( a , b ) ) i n t h e r e g i o n o f s u r v i v a l g r e a t e r t h a n 0 . 2 and l e s s t h a n t h o s e p r e d i c t e d b y e q u a t i o n 3 (S^ l e s s t h a n S ( a , b ) ) i n t h e r e g i o n o f s u r v i v a l l e s s t h a n 0 . 0 5 . T h i s c a n be a t l e a s t p a r t i a l l y e x p l a i n e d by t h e o b s e r v e d d i f f e r e n c e b e t w e e n t h e s u r v i v a l m e a s u r e d by t h e l o w dose a s s a y and t h e c o n v e n t i o n a l a s s a y w h i c h was 0.12±0.08 s t a n d a r d d e v i a t i o n f o r e x p e r i m e n t s e t A and 0.18±.09 s t a n d a r d d e v i a t i o n f o r e x p e r i m e n t s e t B . Thus t h e s u r v i v a l measu red by t h e l o w dose a s s a y was a g a i n m a r g i n a l l y h i g h e r t h a n t h a t measu red by t h e c o n v e n t i o n a l a s s a y . The l o w d o s e a s s a y w o u l d t h e r e f o r e t e n d t o g i v e s u r v i v a l v a l u e s g r e a t e r t h a n t h e v a l u e p r e d i c t e d by e q u a t i o n 3 and t h e c o n v e n t i o n a l a s s a y w o u l d t e n d t o g i v e s u r v i v a l v a l u e s l e s s t h a n t h e p r e d i c t e d v a l u e . T h i s d i s c r e p e n c y i n s u r v i v a l measurements i s s u f f i c i e n t l y s m a l l t h a t i t was i g n o r e d . Q u a n t i t a t i v e s t u d y c o u l d r e v e a l t h e s o u r c e o f t h e d i s c r e p e n c y and p r o v i d e a p p r o p r i a t e c o r r e c t i o n s f o r t h e d a t a . Q u a l i t a t i v e l y t h e d i s c r e p e n c y may be e x p l a i n e d by t h e l a r g e r number o f m a r g i n a l l y v i a b l e c e l l s a t l o w e r s u r v i v a l s , some o f w h i c h were n o t l o c a t e d on t h e l o c a t i o n d a y , ( p o o r l y g r o w i n g c e l l s a r e more d i f f i c u l t t o l o c a t e t h a n t h e h e a l t h y , p r o l i f e r a t i n g c e l l s ) r e s u l t i n g i n a h i g h e r 73 e s t i m a t e o f s u r v i v a l w i t h t h e l o w d o s e a s s a y . P ( X z , v ) i s t h e p r o b a b i l i t y o f o b t a i n i n g a minimum v a l u e o f X 2 = x 2 ( e q u a t i o n 15) i f t h e s u r v i v a l v a l u e s a r e n o r m a l l y d i s t r i b u t e d w i t h mean and s t a n d a r d d e v i a t i o n s d ( S ^ ) . The p a r a m e t e r v i s t h e number o f d e g r e e s o f f r e e d o m . The v a l u e s o f P ( X 2 , v ) f o r t h e r e s u l t s o f e x p e r i m e n t s e t s A and B a r e g i v e n i n t a b l e 3 . E x p e r i m e n t s e t A g i v e s a s e t o f P ( X 2 , v ) w h i c h f a l l r e a s o n a b l y a b o u t t h e e x p e c t e d mean o f 50%. T h i s i s t o be e x p e c t e d i n t h e c a s e o f d a t a s e t s w h i c h c o n t a i n e d more t h a n one e x p e r i m e n t s i n c e t h e s t a n d a r d d e v i a t i o n o f t h e i n d i v i d u a l d a t a p o i n t s was c h o s e n so t h a t t h e r e s i d u a l s ( e q u a t i o n 16) w o u l d be n o r m a l l y d i s t r i b u t e d a b o u t t h e b e s t f i t l i n e a r - q u a d r a t i c dose c u r v e ( e q u a t i o n 1 ) , f o r c i n g t h e sum o f t h e s q u a r e s o f t h e r e s i d u a l s , X 2 t o y i e l d a r e a s o n a b l e v a l u e on t h e a v e r a g e . N o t e t h a t t h r e e o f t h e t e n d a t a s e t s (30%) have P ( X 2 , v ) g r e a t e r t h a n 0 . 9 8 o r l e s s t h a n 0 . 0 2 . T h e r e i s o n l y 4% c h a n c e o f a g i v e n d a t a s e t f a l l i n g o u t s i d e o f t h e s e b o u n d s . The o c c u r e n c e o f t h e s e v e r y l o w and v e r y h i g h v a l u e s o f P ( X 2 , v ) i s an i n d i c a t i o n t h a t t h e t o t a l s t a n d a r d d e v i a t i o n i s n o t p r o p o r t i o n a l t o t h e e s t i m a t e d e x p e r i m e n t a l s t a n d a r d d e v i a t i o n as a s s u m e d , b u t r a t h e r depends on o t h e r u n r e l a t e d f a c t o r s . Thus t h i s a s s u m p t i o n r e s u l t s i n a r a t h e r c r u d e e s t i m a t e o f t h e t o t a l s t a n d a r d d e v i a t i o n , and t h e c o n f i d e n c e c o n t o u r s and P ( X 2 , v ) a r e o n l y r o u g h e s t i m a t e s . N o t e t h a t t h e s u r v i v a l c u r v e s f o r t h e 0 . 0 5 , 1 0 . 0 , and 4 0 . 0 mM m i s o n i d a z o l e c o n c e n t r a t i o n s were o n l y m e a s u r e d o n c e . The s u r v i v a l i n t h e s e c a s e s was n o t s u b j e c t t o ' b i o l o g i c a l v a r i a t i o n ' ( v a r i a t i o n b e t w e e n e x p e r i m e n t s ) and t h e r e f o r e t h e u n c e r t a i n t y was e s t i m a t e d as t h e e s t i m a t e d ' e x p e r i m e n t a l ' u n c e r t a i n t y ( v a r i a t i o n w i t h i n an e x p e r i m e n t where b i o l o g i c a l v a r i a t i o n i s e a s i e r t o c o n t r o l ) . P ( X 2 , v ) = 0 . 2 f o r 5 mM m i s o n i d a z o l e , P ( X 2 , v ) = 0 . 7 f o r 10 mM m i s o n i d a z o l e , and P ( X 2 , v ) = 0 . 2 f o r 40 mM m i s o n i d a z o l e . These a r e r e a s o n a b l e 74 v a l u e s and t h e r e f o r e e q u a t i o n 3 i s a r e a s o n a b l e f i t t o t h i s d a t a . E x p e r i m e n t s e t B g i v e s a s e t o f P ( X 2 , v ) ( t a b l e 3) w h i c h a r e c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t e q u a t i o n 3 i s a s u f f i c i e n t l y good f i t t o ^ t h e d a t a . The r e a s o n a b l e v a l u e s o b t a i n e d f o r P ( X 2 , v ) ( 0 . 2 t o 0 . 8 f o r t h e f i v e d a t a s e t s ) i n d i c a t e s t h a t t h e s t a n d a r d d e v i a t i o n i n t h e d a t a p o i n t s was p r o p e r l y e s t i m a t e d . I n c o m p a r i s o n w i t h e x p e r i m e n t s e t A r e s u l t s , by m a k i n g s e v e r a l measurements o f e a c h d a t a p o i n t a much b e t t e r e s t i m a t e o f t h e t o t a l s t a n d a r d d e v i a t i o n was made. C o n s e q u e n t l y t h e e s t i m a t e d c o n t o u r i n t e r v a l s and P ( X 2 , v ) a r e much more r e l i a b l e t h a n f o r e x p e r i m e n t s e t A . F i g u r e 14 i s a p l o t o f t h e e s t i m a t e d 70% c o n f i d e n c e c o n t o u r s o f t h e p a r a m e t e r s a l p h a and b e t a ( e q u a t i o n 3) f o r e x p e r i m e n t s e t A . The c o n t o u r s t e n d t o be e l l i p t i c a l w i t h t h e i r a x e s o r i e n t e d d i a g o n a l l y w i t h r e s p e c t t o t h e a l p h a and b e t a a x e s d e m o n s t r a t i n g t h e s t r o n g c o r r e l a t i o n b e t w e e n t h e e s t i m a t e s o f a l p h a and b e t a . The a r e a o f t h e e l l i p s e depends d i r e c t l y on t h e e s t i m a t e o f t h e s t a n d a r d d e v i a t i o n o f t h e d a t a p o i n t s , w h i c h was o n l y a r o u g h e s t i m a t e b a s e d on t h e r e s i d u a l s f r o m a l l o f t h e e x p e r i m e n t s o f e x p e r i m e n t s e t s A and B. Thus t h e c o n t o u r s a r e a r o u g h e s t i m a t e o f t h e 70% c o n f i d e n c e l i m i t s . The d e g r e e o f v a r i a t i o n i n t h e s i z e o f t h e c o n t o u r s i s e v i d e n c e o f t h i s . The c o n f i d e n c e c o n t o u r p l o t shows a s i g n i f i c a n t i n c r e a s e i n b e t a f o r c o n c e n t r a t i o n s o f m i s o n i d a z o l e g r e a t e r t h a n 0 . 2 5 mM, w h e r e a s e t h e p l o t shows no s i g n i f i c a n t i n c r e a s e i n a l p h a . F i g u r e s 1 5 , 1 6 , and 17 show t h e e s t i m a t e d 70%, 95%, and 99 .9% c o n f i d e n c e c o n t o u r s r e s p e c t i v e l y f o r e x p e r i m e n t s e t B . Once a g a i n a s t r o n g c o r r e l a t i o n i s o b s e r v e d b e t w e e n t h e e s t i m a t e s o f a l p h a and b e t a . The e s t i m a t e o f t h e s t a n d a r d d e v i a t i o n i n t h e d a t a p o i n t s was o b t a i n e d f r o m measurements o f t h e v a r i a t i o n i n s u r v i v a l a t e a c h d a t a p o i n t (each r a d i a t i o n dose and m i s o n i d a z o l e c o n c e n t r a t i o n ) . T h i s e s t i m a t e w i l l 75 T ! 1 I I I 1 0 . 0 0 0 . 0 5 0 . 1 0 a l p h a ( / G y ) Figure 14: ALPHA AND BETA CONFIDENCE CONTOURS, EXPERIMENT SET A. The 70% confidence l i m i t s of the equation 3 parameters, alpha and beta, were estimated using the chi-squared t e s t . A d i f f e r e n t confidence i n t e r v a l was estimated from the data f o r each concentration of misonidazole as indicated on the graph. Those dataset with a minimum value of chi-squared greater than that which would y i e l d a 70% confidence i n t e r v a l are indic a t e d with a cross (X). 76 CM ' G y 0 . 2 0 ca 0 . 1 0 CD jQ 0 . 0 0 0 . 0 0 0 . 0 5 . 0 . 1 0 a l p h a ( / G y) Figure 15: ALPHA AND BETA 70% CONFIDENCE CONTOURS, EXPERIMENT SET B. The 70% confidence l i m i t s of the equation 3 parameters, alpha and beta, were estimated using the chi-squared t e s t . A d i f f e r e n t confidence i n t e r v a l was estimated from the data f o r each concentration of misonidazole as indicated on the graph. These confidence i n t e r v a l s are much smaller than those estimated for experiment set A, figure 14. Thus the r e s u l t s for experiment set B are much more accurate. 77 CM (3 0 . 2 Oh z °-10 O . O O h 0 . 0 0 0 . 0 5 . 0 . 1 0 a l p h a (/.Gy) Figure 16: ALPHA AND BETA 95% CONFIDENCE CONTOURS EXPERIMENT SET B. The 95% confidence limits of the equation 3 parameters, alpha and beta, were estimated using the chi-squared test. A different confidence interval was estimated from the data for each concentration of misonidazole as indicated on the graph. 78 CM >» CD 0 . 2 0 CO JQ 0 . 0 0 --— » 0 . 0 0 0 . 0 5 . 0 . 1 0 a l p h a ( / G y ) Figure 17: ALPHA AND BETA 99.9% CONFIDENCE CONTOURS EXPERIMENT SET B. The 99 .9% confidence l i m i t s of the equation 3 parameters, alpha and beta, were estimated using the chi-squared t e s t . A d i f f e r e n t confidence i n t e r v a l i s estimated from the data f o r each concentration of misonidazole as indicated on the graph. 79 a p p r o a c h t h e t r u e s t a n d a r d d e v i a t i o n when many measurements a r e made f o r e a c h d a t a p o i n t . Thus t h e c o n f i d e n c e c o n t o u r s a r e r e a s o n a b l e e s t i m a t e s o f t h e t r u e c o n f i d e n c e l i m i t s . The c o n t o u r p l o t shows a s i g n i f i c a n t i n c r e a s e i n b e t a f o r m i s o n i d a z o l e c o n c e n t r a t i o n s g r e a t e r t h a n 0 . 1 mM, w h e r e a s t h e p l o t shows no s i g n i f i c a n t i n c r e a s e i n a l p h a . The r e s u l t s f r o m t h e e a r l i e r e x p e r i m e n t s e t A ( f i g u r e 14) a r e c l e a r l y l e s s a c c u r a t e . B o t h s e t s o f r e s u l t s a r e c o n s i s t e n t w i t h i n t h e e s t i m a t e d e x p e r i m e n t a l u n c e r t a i n t i e s . P l o t s o f t h e most l i k e l y v a l u e s o f a l p h a and b e t a f r o m t a b l e 3 a r e shown i n f i g u r e 18 ( e x p e r i m e n t s e t A) and f i g u r e 19 ( e x p e r i m e n t s e t B) w i t h t h e 70% c o n f i d e n c e i n t e r v a l . From t h e s e f i g u r e s t h e a v e r a g e s o f a l p h a and b e t a i n v a r i o u s r a n g e s o f m i s o n i d a z o l e c o n c e n t r a t i o n s were e s t i m a t e d . The r e s u l t s a r e shown i n t a b l e I V . The two s e t s o f r e s u l t s a r e c l e a r l y c o n s i s t e n t w i t h e a c h o t h e r w i t h i n t h e e s t i m a t e d e x p e r i m e n t a l u n c e r t a i n t i e s . The r e s u l t s f o r e x p e r i m e n t s e t B w e r e o b t a i n e d f r o m f e w e r e x p e r i m e n t s and a r e o f g r e a t e r a c c u r a c y due t o i m p r o v e m e n t s i n t h e e x p e r i m e n t a l method as d i s c u s s e d i n s e c t i o n 3 . 3 . 3 . The r e s u l t s o b t a i n e d f o r a l p h a show t h a t a l p h a d o e s n o t change s i g n i f i c a n t l y b u t r e m a i n s c o n s t a n t w i t h i n e x p e r i m e n t a l u n c e r t a i n t y . B e t a c l e a r l y i n c r e a s e s i n t h e i n t e r v a l o f m i s o n i d a z o l e c o n c e n t r a t i o n b e t w e e n 0 . 1 mM and 5 . 0 mM. The n a t u r e o r u p p e r bound o f t h i s i n c r e a s e was n o t a c c u r a t e l y measu red by t h e s e e x p e r i m e n t s . More h i g h dose p o i n t s and more c o n c e n t r a t i o n s o f m i s o n i d a z o l e w i t h i n t h i s r a n g e a r e r e q u i r e d . 4 . 3 SIGNIFICANCE OF THE RESULTS. Two s e t s o f e x p e r i m e n t s , A and B, were p e r f o r m e d c o n s e c u t i v e l y t o measure t h e s u r v i v a l o f CHO c e l l s s u b j e c t e d t o v a r i o u s d o s e s o f X - r a d i a t i o n and r a d i o s e n s i t i z e r c o n c e n t r a t i o n s . E x p e r i m e n t s e t A p r o v i d e d p r e l i m i n a r y 80 Figure 1 8 : ALPHA AND BETA DEPENDENCE ON MISONIDAZOLE CONCENTRATION, EXPERIMENT SET A. Survival curves were obtained at various concentrations of the radiosensitizer misonidazole. The parameters alpha and beta of the linear-quadratic equation 3 were estimated for each curve. The estimated 70% confidence limits from figure 14 are shown. 81 F i g u r e 1 9 : ALPHA AND BETA DEPENDENCE ON MISONIDAZOLE CONCENTRATION, EXPERIMENT SET B. A l p h a and b e t a were e s t i m a t e d as i n f i g u r e 18 . The 70% c o n f i d e n c e l i m i t s from f i g u r e 16 a r e shown. These d a t a a r e c l e a r l y much more r e l i a b l e than t h e experiment s e t A d a t a o f f i g u r e 1 8 . Beta c l e a r l y i n c r e a s e s w i t h m i s o n i d a z o l e c o n c e n t r a t i o n , a l p h a remains c o n s t a n t w i t h i n the e x p e r i m e n t a l u n c e r t a i n t y . 82 TABLE IV T a b l e I V : THE RANGE OF THE PARAMETERS ALPHA AND BETA. The r a n g e o f t h e p a r a m e t e r s a l p h a and b e t a as e s t i m a t e d by c h i - s q u a r e d m i n i m i z a t i o n , e x p e r i m e n t s e t s A and B . p a r a m e t e r [MISONIDAZOLE] E x p e r i m e n t s e t A E x p e r i m e n t s e t B a l p h a a l l c o n c e n t r a t i o n s 0 . 0 4 2 1 0 . 0 1 0 G y " 1 0 . 0 3 9 1 0 . 0 1 0 G y " 1 b e t a 0 - 0 . 1 mM 0 . 0 0 2 3 1 0 . 0 0 0 3 G y " 2 0 . 0 0 1 9 1 0 . 0 0 0 2 G y " 2 - 2 -2 b e t a 5 . 0 - 4 0 mM 0 . 0 0 6 5 1 0 . 0 0 5 6 Gy 0 . 0 1 1 8 1 0 . 0 0 2 4 Gy 83 d a t a w h i c h h a s n o t b e e n t a b u l a t e d h e r e . The s u r v i v a l was n o t a v e r a g e d . The s u r v i v a l d a t a f o r e x p e r i m e n t s e t B i s t a b u l a t e d i n t a b l e I I . The s u r v i v a l v a l u e s f o r e a c h dose and m i s o n i d a z o l e c o n c e n t r a t i o n were a v e r a g e d o v e r t h e t h r e e e x p e r i m e n t s p e r f o r m e d f o r e x p e r i m e n t s e t B. A l i n e a r q u a d r a t i c m o d e l ( e q u a t i o n 3) was f i t t o t h e d a t a o f b o t h e x p e r i m e n t s e t s t o e s t i m a t e t h e p a r a m e t e r s a l p h a and b e t a . R e s u l t s a r e t a b u l a t e d i n t a b l e I I I and t a b l e I V and g r a p h e d i n f i g u r e s 14 t o 19 . The ' b e s t ' e s t i m a t e s o f t h e s u r v i v a l c u r v e p a r a m e t e r s f o r t h e e x p e r i m e n t s e t B d a t a show a s i g n i f i c a n t t r e n d i n t h e h i g h - d o s e s u r v i v a l - 2 p a r a m e t e r , b e t a , w h i c h i n c r e a s e f r o m 0.0019±0.0002 Gy t o - 2 0.012±0.002 Gy b e t w e e n m i s o n i d a z o l e c o n c e n t r a t i o n s o f 0 . 1 and 5 . 0 mM. U n c e r t a i n t i e s a r e e s t i m a t e s o f t h e maximum r a n g e o f b e t a w i t h i n 70% c o n f i d e n c e . T h e r e i s no s i g n i f i c a n t t r e n d i n t h e l o w dose s u r v i v a l p a r a m e t e r , a l p h a , w h i c h r e m a i n s c o n s t a n t a t 0.039±0.010 Gy ^ w i t h i n 70% c o n f i d e n c e . The s u r v i v a l d a t a w i t h t h e s e b e s t f i t c u r v e s a r e p l o t t e d a g a i n s t d o s e i n f i g u r e 20 . A t l o w d o s e s i t i s n o t p o s s i b l e t o d i s t i n g u i s h b e t w e e n t h e d a t a p o i n t s o b t a i n e d w i t h d i f f e r e n t c o n c e n t r a t i o n s o f m i s o n i d a z o l e . 4 . 3 . 1 MISONIDAZOLE DMF dependence on s u r v i v a l l e v e l . F rom t h e ' b e s t ' e s t i m a t e s o f a l p h a and b e t a f r o m e x p e r i m e n t s e t B DMF's o f t h e v a r i o u s c o n c e n t r a t i o n s o f m i s o n i d a z o l e were c a l c u l a t e d a t s u r v i v a l l e v e l s o f 0 . 0 1 t o 0 . 8 . I n t a b l e V t h e s e r e s u l t s a r e compared t o v a l u e s o b t a i n e d f r o m t h e s u r v i v a l c u r v e s o f Chinese h a m s t e r c e l l s t r e a t e d w i t h m i s o n i d a z o l e and X - r a y s b y Moore e t a l , 1 9 7 6 , and M c N a l l y , 1976 . L i k e most s u r v i v a l d a t a i n t h e l i t e r a t u r e t h e d a t a o f b o t h Moore and M c N a l l y were o b t a i n e d f r o m s u r v i v a l measurements a t s u r v i v a l l e v e l s u n d e r 0 . 5 . A l l o f t h e r e s u l t s a r e 8 4 0.0 2.0 4.0 6.0 o o s <z cs "Y* •> F i g u r e 2 0 : SURVIVAL CURVES, EXPERIMENT SET B. The c o n c e n t r a t i o n o f m i s o n i d a z o l e f o r the d a t a p o i n t s and s u r v i v a l c u r v e s a r e as f o l l o w s : 1- 0 mM, 2- 0 . 1 mM, 3- 0 . 4 mM, 4 - 1 . 5 mM, and 5- 5 . 0 mM m i s o n i d a z o l e . The drug was p r e s e n t d u r i n g i r r a d i a t i o n t o measure i t s r a d i o s e n s i t i z i n g a b i l i t y . The lower graph i s a m a g n i f i e d view o f t h e d a t a shown i n t h e upper graph i n , the r e g i o n 0 - 7 . 5 Gray. 85 TABLE V T a b l e V : DMF OF 0 - 5 . 0 mM MISONIDAZOLE AT SURVIVAL LEVELS OF 0 . 0 1 TO 0 . 8 , EXPERIMENT SET B . The DMF o f v a r i o u s c o n c e n t r a t i o n s o f m i s o n i d a z o l e as c a l c u l a t e d f r o m t h e r e s u l t s o f e x p e r i m e n t s e t B a r e l i s t e d . DMF was c a l c u l a t e d as t h e d o s e r e q u i r e d a t 0 mM m i s o n i d a z o l e t o r e s u l t i n t h e g i v e n s u r v i v a l d i v i d e d by t h e dose r e q u i r e d a t t h e g i v e n c o n c e n t r a t i o n s o f m i s o n i d a z o l e ( [MISO]) t o r e s u l t i n t h e same s u r v i v a l . The u n c e r t a i n t i e s a r e t h e 70% c o n f i d e n c e i n t e r v a l s . R e s u l t s f r o m Moore e t a l (1976) and M c N a l l y (1976) a r e l i s t e d f o r c o m p a r i s o n . [MISO] DMF a t S=0.8 DMF a t S=0.5 DMF a t S=0.1 DMF a t S=0 .01 (mM) FROM EXPERIMENT SET B: 0 . 1 1.0±0.4 1 . 0±0.4 1 . 0 1 0 . 4 1 . 1 1 0 . 3 0 . 4 1.1±0.4 1 . 2±0.4 - 1 . 3 1 0 . 4 1 . 4 1 0 . 4 1 .5 1.3±0.4 1 . 5±0.3 1 . 8 1 0 . 3 1 . 9 1 0 . 3 5 . 0 1.4±0.4 1 . 7 1 0 . 4 2 . 1 1 0 . 4 2 . 3 1 0 . 3 FROM MOORE e t a l ( 1 9 7 6 ) : 0 . 1 1 . 2 1 . 3 1 .0 1 .8 1 . 8 15 2 . 0 2 . 3 FROM MCNALLY ( 1 9 7 6 ) : 5 . 0 2 . 1 2 . 3 86 c o n s i s t e n t . Thus a l p h a need n o t i n c r e a s e t o f i t t h e l i n e a r - q u a d r a t i c e q u a t i o n t o t h e m e a s u r e d s u r v i v a l v a l u e s a t h i g h c o n c e n t r a t i o n o f m i s o n i d a z o l e ( 5 - 1 5 mM). P a l c i c e t a l (1982) h a v e r e c e n t l y m e a s u r e d t h e OER f o r a s y n c h r o n o u s CHO c e l l s u s i n g a p r o c e d u r e s i m i l a r t o t h e one r e p o r t e d h e r e . They f o u n d t h r o u g h a m o d e l - i n d e p e n d e n t a n a l y s i s t h a t t h e OER d e c r e a s e s f r o m 2 . 8 a t 0 . 0 1 s u r v i v a l t o a b o u t 1 .7 a t s u r v i v a l l e v e l s g r e a t e r t h a n 0 . 7 as shown i n f i g u r e s 1 and 2 . P a r t o f t h e m o t i v a t i o n f o r t h i s s t u d y was t o d e t e r m i n e i f t h e m i s o n i d a z o l e DMF a l s o d e c r e a s e s a t h i g h s u r v i v a l l e v e l s . T h i s h a s now b e e n c l e a r l y d e m o n s t r a t e d f o r t h e s e t o f e x p e r i m e n t a l c o n d i t i o n s u s e d i n t h i s s t u d y . T h i s r e s u l t was a n t i c i p a t e d s i n c e t h e mechan ism o f m i s o n i d a z o l e r a d i o s e n s i t i z a t i o n i s c o n s i d e r e d by many t o be a n a l a g o u s t o t h e mechan ism o f o x y g e n r a d i o s e n s i t i z a t i o n (Adams, 1973 ; Wardman, 1 9 7 7 ) . 4 . 3 . 2 C l i n i c a l s i g n i f i c a n c e o f t h e r e s u l t s . The d a t a o f e x p e r i m e n t s e t B g i v e s t h e most a c c u r a t e r e s u l t s . F i g u r e 15 shows t h a t t h e p a r a m e t e r a l p h a d o e s n o t change s i g n i f i c a n t l y i n t h e m i s o n i d a z o l e c o n c e n t r a t i o n r a n g e o f 0 - 5 . 0 mM and t h a t b e t a does n o t change s i g n i f i c a n t l y b e t w e e n 0 mM and 0 . 1 mM m i s o n i d a z o l e . T h e r e f o r e no s i g n i f i c a n t d e c r e a s e i n c e l l s u r v i v a l i s o b s e r v e d i n t h e m i s o n i d a z o l e c o n c e n t r a t i o n r a n g e 0 - 0 . 1 mM and o n l y b e t a i s o b s e r v e d t o i n c r e a s e a t m i s o n i d a z o l e c o n c e n t r a t i o n s g r e a t e r t h a n o r e q u a l t o 0 . 4 mM. I t c a n be c a l c u l a t e d t h a t a t t h e dose o f 2 Gy i n h y p o x i a , a p p r o x i m a t e l y 20% o f a l l c e l l s a r e i n a c t i v a t e d , 90% by t h e l i n e a r c o e f f i c i e n t a l p h a , and o n l y 10% by t h e q u a d r a t i c c o e f f i c i e n t b e t a . I f a d r u g l i k e m i s o n i d a z o l e c a n n o t s i g n i f i c a n t l y enhance tumour c e l l i n a c t i v a t i o n by t h e a l p h a t e r m , t h e n one c a n n o t s i g n i f i c a n t l y i m p r o v e r a d i o t h e r a p y i n f r a c t i o n a t e d r e g i m e s where f r a c t i o n s a r e s m a l l . The u s e o f l a r g e r d o s e f r a c t i o n s t o make t h e d r u g e f f e c t i v e c o u l d i n c r e a s e t h e 87 r a d i o s e n s i t i z a t i o n , p r o v i d e d t h a t s u c h a r e g i m e i s n o t a d i s a d v a n t a g e i n o t h e r r e s p e c t s i n c o m p a r i s o n t o a r e g i m e where s m a l l e r f r a c t i o n s a r e e m p l o y e d . These o b s e r v a t i o n s a r e r e f l e c t e d i n t h e s u r v i v a l c u r v e s shown i n f i g u r e 20 . I t i s i m p o s s i b l e t o d i s t i n g u i s h b e t w e e n t h e s u r v i v a l measurements and t h e b e s t f i t s u r v i v a l c u r v e s a t d o s e s o f r a d i a t i o n l e s s t h a n a p p r o x i m a t e l y 3 G r a y . M i s o n i d a z o l e may s t i l l p r o v i d e a c l i n i c a l g a i n due t o t h e c y t o t o x i c i t y o f t h e d r u g and t h e enhancement o f t h e c y t o t o x i c i t y by r a d i a t i o n . The dose m o d i f y i n g f a c t o r (DMF) o f m i s o n i d a z o l e h a s b e e n e s t i m a t e d f o r t h e d a t a o f e x p e r i m e n t s e t B and t a b u l a t e d i n t a b l e I V . The DMF i s n o t s i g n i f i c a n t l y d i f f e r e n t f r o m u n i t y f o r 0 . 1 mM m i s o n i d a z o l e f o r d o s e s a s h i g h a s 30 G r a y , f o r 0 . 4 mM f o r d o s e s as h i g h a s 12 G r a y , f o r 1 .5 mM f o r d o s e s as h i g h as a b o u t 3 G r a y , and f o r 5 . 0 mM f o r d o s e s as h i g h as a b o u t 2 G r a y . The maximum DMF i s 2 . 1 1 0 . 5 . T h i s o c c u r s f o r 5 . 0 mM m i s o n i d a z o l e a t a s u r v i v a l o f 0 . 0 1 c o r r e s p o n d i n g t o a dose o f 12 Gy . O t h e r r e s e a r c h e r s have m e a s u r e d t h e DMF o f m i s o n i d a z o l e i n t h e same e x p e r i m e n t a l s y s t e m b u t o n l y a t s u r v i v a l l e v e l s l e s s t h a n 0 . 5 . The r e s u l t s f r o m two i n v e s t i g a t o r s a r e t a b u l a t e d a l o n g w i t h t h e r e s u l t s o f t h i s s t u d y t o show t h a t t h e y a r e c o n s i s t e n t . The e x p e r i m e n t shows c l e a r l y t h a t , w i t h i n r e a s o n a b l e e x p e r i m e n t a l e r r o r , t h e r a d i o s e n s i t i z e r f a i l s t o d e c r e a s e c e l l s u r v i v a l a t c l i n i c a l l y a c c e p t a b l e r a d i a t i o n d o s e s and m i s o n i d a z o l e c o n c e n t r a t i o n s . 4 . 3 . 3 R a d i o b i o l o g i c a l s i g n i f i c a n c e . A c c o r d i n g t o t h e r e s u l t s o f t h e c h i - s q u a r e t e s t ( t a b l e I I I ) t h e l i n e a r - q u a d r a t i c mode l ( e q u a t i o n 3) i s a r e a s o n a b l e f i t t o t h e s u r v i v a l d a t a . F u r t h e r m o r e , m i s o n i d a z o l e h a s no s i g n i f i c a n t e f f e c t on t h e a l p h a component and c a u s e s t h e b e t a component t o i n c r e a s e f o r m i s o n i d a z o l e c o n c e n t r a t i o n s o f 0 . 4 mM and g r e a t e r . The 88 m o l e c u l a r t h e o r y o f c e l l s u r v i v a l o f C h a d w i c k and L e e n h o u t s (1981) p r o p o s e s t h a t a l p h a i s t h e one h i t component o f c e l l i n a c t i v a t i o n and b e t a i s t h e m u l t i p l e h i t component . I f m i s o n i d a z o l e o n l y m e a s u r a b l y a f f e c t s t h e b e t a component t h e n i t may o n l y i n t e r a c t o n l y w i t h t h e m u l t i p l e h i t component o f c e l l i n a c t i v a t i o n . F u r t h e r s t u d y i n t o t h e s i g n i f i c a n c e o f t h i s r e s u l t w i l l be l e f t t o f u t u r e r e s e a r c h . I t i s i m p o r t a n t t o a c k n o w l e d g e t h a t t h e s e s u r v i v a l measurements c a n be f i t e q u a l l y w e l l by s e v e r a l ( i f n o t most ) s u r v i v a l m o d e l s i n t h e l i t e r a t u r e , as d i s c u s s e d i n s e c t i o n 3 . 1 . The c h o i c e o f e q u a t i o n 3 t o f i t t h e d a t a does n o t c o n s t i t u t e a b i a s t o w a r d t h e m o l e c u l a r t h e o r y ; i t i s s i m p l y one o f t h e l e a s t c o m p l e x m a t h e m a t i c a l e q u a t i o n s w h i c h c a n a d e q u a t e l y f i t t h e d a t a and c a n be r e a d i l y i n t e r p r e t e d w i t h r e s p e c t t o t h e dependence on r a d i a t i o n dose o f t h e e f f e c t o f m i s o n i d a z o l e on c e l l s u r v i v a l . 4 . 3 . 4 S u g g e s t e d i m p r o v e m e n t s t o t h e e x p e r i m e n t a l m e t h o d . The i m p r o v e m e n t s t o t h e e x p e r i m e n t a l p r o c e d u r e o f e x p e r i m e n t s e t B r e s u l t e d i n a more a c c u r a t e and e a s i e r method t o o b t a i n d a t a . The f o l l o w i n g i m p r o v e m e n t s s h o u l d be c o n s i d e r e d f o r f u t u r e e x p e r i m e n t s : 1 . Improve t h e c o r r e l a t i o n b e t w e e n t h e c e l l s u r v i v a l measu red by t h e l o w d o s e and c o n v e n t i o n a l a s s a y s . The m a j o r s o u r c e o f d i s c r e p e n c y i s t h e b i a s i n s e l e c t i n g v i a b l e c e l l s on l o c a t i o n day f o r t h e l o w dose a s s a y . T h i s b i a s c a n be s i g n i f i c a n t l y r e d u c e d by l o c a t i n g c e l l s d u r i n g t h e f i r s t 24 h o u r s a f t e r i r r a d i a t i o n . T h i s i s a p r e d i c t i o n b a s e d on q u a l i t a t i v e a n a l y s i s o f t h e t i m e c o u r s e o f m a c r o s c o p i c e v e n t s ( f o r m a t i o n o f g i a n t c e l l s , s h r i v e l l e d c e l l s , e t c . ) a f t e r c e l l s t r e a t e d w i t h m i s o n i d a z o l e a r e e x p o s e d t o X - i r r a d i a t i o n . 89 2 . Use t h e l o w d o s e a s s a y o n l y t o measure s u r v i v a l s g r e a t e r t h a n 0 . 5 . O t h e r w i s e , i t i s e a s i e r t o m e a s u r e s u r v i v a l w i t h t h e c o n v e n t i o n a l a s s a y t o t h e same d e g r e e o f a c c u r a c y . 3 . Choose d o s e p o i n t s f o r s u r v i v a l e s t i m a t e s i n s u c h a way as t o o b t a i n e v e n i n c r e m e n t s i n t h e l o g a r i t h m o f t h e s u r v i v a l . Such d a t a i s w e l l s u i t e d t o f i t s t o s u r v i v a l m o d e l s . N o t e t h a t t h e h i g h d o s e p o i n t s a r e as i m p o r t a n t a s t h e l o w d o s e p o i n t s f o r e s t i m a t i n g s u r v i v a l m o d e l p a r a m e t e r s . 4 . O b t a i n a t l e a s t t h r e e s u r v i v a l measurements f o r e a c h r a d i a t i o n dose and m i s o n i d a z o l e c o n c e n t r a t i o n i n o r d e r t o o b t a i n a r e a s o n a b l e e s t i m a t e o f t h e mean and s t a n d a r d d e v i a t i o n o f t h e s u r v i v a l . Use s y n c h r o n o u s c e l l s i n o r d e r t o r e d u c e t h e v a r i a t i o n b e t w e e n e x p e r i m e n t s . 5 . When t e c h n o l o g y p e r m i t s , t h e r e l i a b i l i t y o f t h e c o n v e n t i o n a l a s s a y c a n be i n c r e a s e d t o a p p r o a c h t h a t o f t h e l o w d o s e a s s a y by u s i n g a c e l l s o r t e r w h i c h p l a t e s c e l l s a f t e r c o u n t i n g them. T h i s w i l l i n c r e a s e t h e p l a t i n g e f f i c i e n c y i f l e s s d e b r i s i s c o u n t e d a s c e l l s and i t w i l l a l s o i n c r e a s e t h e r e l i a b i l i t y o f t h e number o f c e l l s p l a t e d . I f p l a t e d c e l l s a r e t h e n c l a s s i f i e d w i t h t h e m i c r o s c o p e ( p e r h a p s a u t o m a t i c a l l y by compute r ) t h e c o n v e n t i o n a l a s s a y c o u l d a p p r o a c h t h e r e l i a b i l i t y o f t h e l o w d o s e a s s a y w i t h o u t r e q u i r i n g t h e t i m e c o n s u m i n g l o c a t i o n o f c e l l s u n d e r t h e m i c r o s c o p e . A n o t h e r method o r r e l i e v i n g t h e e x p e r i m e n t e r o f t h e t e d i o u s t a s k o f l o c a t i n g c e l l s i s t o add a TV d i g i t i z e r t o t h e ALDAS c o m p u t e r and t o t e a c h t h e c o m p u t e r t o r e c o g n i z e c e l l s f r o m d i g i t i z e d TV p i c t u r e s . T h i s p r o c e d u r e w o u l d h a v e a l l o f t h e s t a t i s t i c a l a d v a n t a g e s o f t h e l o w d o s e a s s a y a n d , i n a d d i t i o n , w o u l d be much f a s t e r t h a n t h e c u r r e n t p r o c e d u r e . 90 4.4 SURVIVAL MEASUREMENTS AT LOW DOSES. The low dose assay was used to measure loss of c e l l p r o l i f e r a t i v e capacity f o r s u r v i v a l s of greater than 50% of the plated c e l l s . The automated low dose assay system (ALDAS) reduced assay time and experimenter s t r a i n r e s u l t i n g i n a two- to th r e e - f o l d increase i n p r o d u c t i v i t y and reduced experimental uncertainty. Using ALDAS, the experimenter can locate about ten c e l l s per minute or 150 c e l l s i n 15 minutes. These figures assume a p l a t i n g of about 0.2 cells/mm 2 and a f i e l d of view of about 1 mm2 when lo c a t i n g c e l l s two to three days a f t e r i r r a d i a t i o n . Lower p l a t i n g d e n s i t i e s , a smaller f i e l d of view, and e a r l i e r c e l l l o c a t i o n w i l l a l l tend to decrease the speed with which c e l l s can be located. The experimenter can locate c e l l s f o r up to four hours per day, (approximately 16 plated v e s s e l s , l o c a t i n g 150 c e l l s per vessel) f o r about three consecutive days before becoming overstressed and making s i g n i f i c a n t observational e r r o r s . This could be greatly improved by further automating ALDAS (section 4.3.4). It has been found that the system c o n s i s t e n t l y meets the s p e c i f i c a t i o n s l i s t e d i n table VI. The i n t e r a c t i v e Fortran programs developed f o r the LDA system provide necessary and s u f f i c i e n t options, v a r i a b l e program parameters, and user selected a l t e r n a t i v e s to give the optimum balance between software f l e x i b i l i t y and ease of use. The programs are well documented and can be e a s i l y updated or modified f o r unforseen a p p l i c a t i o n s . Some readers may be interest e d i n acquiring there own ALDAS. A system of these s p e c i f i c a t i o n s i s curr e n t l y not stocked by any manufacturer. To b u i l d and t e s t the system requires the service of one computer technician 91 TABLE V I T a b l e V I : SPECIFICATIONS FOR THE AUTOMATED LOW DOSE ASSAY, ALDAS: S t e p s i z e : 10 um S c a n d i m e n s i o n s : 7 cm X 12 cm R e p e a t a b i l i t y : 100 um Minimum r e p e a t a b i l i t y : l e s s t h a n 10 um i f l o c a t i o n s a r e a l w a y s a p p r o a c h e d f r o m t h e same d i r e c t i o n . Maximum s p e e d , ASCAN: 6000 s t e p s / s e c . s o f t w a r e a d j u s t a b l e . (At h i g h e r s p e e d s may g e t m i s s e d s t e p s a t edge o f s c a n o r i f t h e l o a d i s t o o h e a v y . ) Maximum s p e e d , MSCAN: 5000 s t e p s / s e c . s o f t w a r e a d j u s t a b l e . (At h i g h e r s p e e d s may g e t m i s s e d s t e p s a t edge o f s c a n o r i f t h e l o a d i s t o o h e a v y . ) 92 ( t o b u i l d t h e i n t e r f a c e b e t w e e n t h e c o m p u t e r and t h e s t e p p i n g m o t o r s ) , one m a c h i n i s t ( t o b u i l d t h e s t a g e ) , and a c o m p u t e r programmer f o r one t o two m o n t h s . I t w o u l d t a k e a b o u t two t o f o u r weeks f o r an e x p e r i m e n t e r t o become p r o f i c i e n t a t u s i n g t h e l o w dose a s s a y a l o n g w i t h ALDAS. R e s e a r c h i n t o t h e e f f e c t s o f l o w d o s e s o f i o n i z i n g r a d i a t i o n on c e l l s u r v i v a l s h o u l d be e n c o u r a g e d . The r e s u l t s o f t h i s s t u d y f o r m i s o n i d a z o l e r a d i o s e n s i t i z a t i o n , e x e m p l i f y t h e need f o r d i r e c t measurements a t l o w d o s e s . 4 . 5 FUTURE RESEARCH 4 . 5 . 1 D i s c r e p e n c y i n s u r v i v a l measurements b e t w e e n a s s a y s . The l o w dose a s s a y t e n d e d t o y i e l d s l i g h t l y h i g h e r v a l u e s o f s u r v i v a l t h a n t h e h i g h dose a s s a y as n o t e d i n s e c t i o n 4 . 2 . 3 . The a v e r a g e d i f f e r e n c e i n s u r v i v a l i s 0 . 1 2 + 0 . 1 1 f o r e x p e r i m e n t s e t A and 0.18±0.12 f o r e x p e r i m e n t s e t B. These c o m p a r i s o n s were made a t a s u r v i v a l l e v e l o f a b o u t 50% and c a n q u a l i t a t i v e l y be e x p l a i n e d by t h e e f f e c t o f m i s o n i d a z o l e and X - r a d i a t i o n on t h e d e v e l o p e m e n t o f a c e l l i n t o a c o l o n y ; t h a t i s , a p r o p o r t i o n o f t h e p l a t e d c e l l s became n o n - v i a b l e w i t h i n two t o t h r e e d a y s a f t e r i r r a d i a t i o n and w e r e n o t c o u n t e d i n t h e l o w d o s e a s s a y when t h e c e l l s were l o c a t e d . More d e t a i l e d r e s e a r c h i s r e q u i r e d t o c l e a r l y d e m o n s t r a t e t h i s ; f o r e x a m p l e , one c o u l d f o l l o w t h e g r o w t h o f c e l l s p l a t e d f o r t h e l o w dose a s s a y and d e t e r m i n e t h e r e l a t i v e number o f c e l l s t h a t become m a c r o s c o p i c a l l y n o n - v i a b l e i n s a y o n e - h a l f day i n t e r v a l s f r o m t h e t i m e o f i r r a d i a t i o n and measure how t h i s r e l a t i v e number depends on t h e r e s u l t i n g s u r v i v a l . T h i s e x p e r i m e n t c o u l d y i e l d a c o r r e c t i o n f a c t o r f o r s u r v i v a l d e p e n d i n g on t h e day a f t e r i r r a d i a t i o n i n w h i c h c e l l s a r e l o c a t e d . I t c o u l d a l s o p r o v i d e a more q u a n t i t a t i v e measure o f t h e maximum t i m e w h i c h s h o u l d e l a p s e b e t w e e n 93 c e l l i r r a d i a t i o n and c e l l l o c a t i o n . 4 . 5 . 2 S u r v i v a l dependence on o x y g e n t e n s i o n . T h i s e x p e r i m e n t h a s 3 shown t h a t f o r an o x y g e n t e n s i o n o f 10 ppm (0.1%) t h e r e i s no m e a s u r a b l e d e c r e a s e i n c e l l s u r v i v a l a t l o w d o s e s o f i r r a d i a t i o n ( 0 - 2 G r a y ) i n t h e p r e s e n c e o f 0 - 5 mM o f t h e r a d i o s e n s i t i z e r m i s o n i d a z o l e . I t may be o f v a l u e t o p e r f o r m t h e s e measurements a t o t h e r o x y g e n t e n s i o n s i n t h a t f o r t h e e x p e r i m e n t a l c o n d i t i o n s u s e d i n t h i s t h e s i s t h e r e may be s u f f i c i e n t o x y g e n p r e s e n t a t l o w d o s e s o f i r r a d i a t i o n t o mask t h e e f f e c t o f t h e r a d i o s e n s i t i z e r . 4 . 5 . 3 O b s e r v a t i o n o f c e l l u l a r g r o w t h and d i v i s i o n . The l o w dose a s s a y w i t h ALDAS i s an i d e a l s y s t e m f o r s t u d y i n g t h e p r o g r e s s o f X - i r r a d i a t e d mammal ian c e l l s . P a t t e r n s o f g r o w t h c o u l d be d e s c r i b e d and t h e dependenc o f t h e p r o p o r t i o n o f c e l l s w h i c h f o l l o w t h e s e p a t t e r n s on r a d i a t i o n d o s e , d r u g c o n c e n t r a t i o n , o r o t h e r e n v i r o n m e n t a l f a c t o r s c o u l d b e m e a s u r e d . R e s e a r c h o f t h i s t y p e w o u l d l e a d t o a r e f i n e d u n d e r s t a n d i n g o f t h e i n t e r a c t i o n o f r a d i a t i o n w i t h c e l l s . S u c h work h a s a l r e a d y begun i n o u r own l a b a s w e l l a s o t h e r l a b s . (Colombo and M a r i n , 1963 ; S a k k a e t a l , 1975 ; G r o t e e t a l , 1981a and 1 9 8 1 b . ) . 4 . 5 . 4 S u r v i v a l measurements a t l o w d o s e s o f i o n i z i n g r a d i a t i o n . C e l l s u r v i v a l e f f e c t s w h i c h have b e e n m e a s u r e d a t h i g h d o s e s o f r a d i a t i o n c a n be measu red a t l o w d o s e s . These e f f e c t s i n c l u d e t h e e f f e c t s o f L E T , t h e p r e s e n c e (or a b s e n c e ) o f r a d i o s e n s i t i z e r s , r a d i o p r o t e c t o r s and o t h e r a g e n t s , t h e l o c a t i o n o f t h e c e l l s i n t h e c e l l c y c l e , e t c . S i n c e i t i s n o t c l e a r how measurements o b t a i n e d a t h i g h e r d o s e s o f r a d i a t i o n c a n be e x t r a p o l a t e d t o l o w e r d o s e s i t w o u l d be u s e f u l t o make measurements a t l o w d o s e s . The r e s u l t s c o u l d have i m p o r t a n t c l i n i c a l i m p l i c a t i o n s and may s h e d some l i g h t on b a s i c r a d i o b i o l o g i c a l q u e s t i o n s . 94 4 . 6 SUMMARY. The p u r p o s e o f t h i s s t u d y was t o measure t h e r a d i o s e n s i t i z i n g a b i l i t y o f m i s o n i d a z o l e a t l o w d o s e s o f r a d i a t i o n . To a c c o m p l i s h t h i s i t was n e c e s s a r y t o i m p r o v e t h e u n c e r t a i n t y o f t h e c o n v e n t i o n a l c e l l s u r v i v a l a s s a y by l o c a t i n g i n d i v i d u a l c e l l s i m m e d i a t e l y a f t e r i r r a d i a t i o n and f o l l o w i n g t h e i r g r o w t h u s i n g a m i c r o s c o p e . W i t h t h i s improvement t h e u n c e r t a i n t y i n s u r v i v a l was r e d u c e d t o 3-10% a t s u r v i v a l l e v e l s o f 50% o r m o r e . A s y s t e m i n c o r p o r a t i n g a c o m p u t e r - c o n t r o l l e d s t e p p i n g - m o t o r - d r i v e n m i c r o s c o p e s t a g e was d e s i g n e d and b u i l t t o p r o v i d e f o r e a s e i n c e l l l o c a t i o n and c l a s s i f i c a t i o n . R e s u l t s were e a s i l y o b t a i n e d u s i n g t h i s s y s t e m and were much more a c c u r a t e t h a n r e s u l t s o b t a i n e d m a n u a l l y . W i t h t h e a u t o m a t e d s y s t e m a l l o f t h e measurements r e q u i r e d t o a s s e s s t h e e f f e c t o f f i v e d i f f e r e n t c o n c e n t r a t i o n s o f m i s o n i d a z o l e ( i n c l u d i n g t h e c o n t r o l w i t h no m i s o n i d a z o l e ) c o u l d be made i n one e x p e r i m e n t . R e p e t i t i o n o f t h e same e x p e r i m e n t g r e a t l y i m p r o v e d t h e s u r v i v a l u n c e r t a i n t y be i m p r o v i n g s t a t i s t i c s and r e d u c i n g t h e e r r o r s due t o v a r i a t i o n s b e t w e e n e x p e r i m e n t s . The l i n e a r - q u a d r a t i c e q u a t i o n 3 was f i t t o t h e s u r v i v a l d a t a . The h i g h d o s e p a r a m e t e r , b e t a , v a r i e d s i g n i f i c a n t l y i n t h e m i s o n i d a z o l e - 2 c o n c e n t r a t i o n r a n g e 0 . 1 t o 5 . 0 mM f r o m 0 . 0 0 1 9 + 0 . 0 0 0 2 t o 0 . 0 1 2 1 0 . 0 0 2 Gy . The l o w dose p a r a m e t e r , a l p h a , r e m a i n e d c o n s t a n t a t 0 . 0 3 9 1 0 . 0 1 0 Gy \ Thus a l t h o u g h m i s o n i d a z o l e r a d i o s e n s i t i z e s c e l l s t o h i g h d o s e s o f r a d i a t i o n , t h e s e r e s u l t s show a m i n i m a l r a d i o s e n s i t i z a t i o n a t c l i n i c a l ( low) d o s e s o f r a d i a t i o n . T h i s may e x p l a i n t h e m i n i m a l s u c c e s s o f m i s o n i d a z o l e i n p a t i e n t t r e a t m e n t s t o d a t e . T h i s s t u d y e x e m p l i f i e s t h e v a l u e o f m e a s u r i n g c e l l s u r v i v a l a t l o w d o s e s o f r a d i a t i o n . 95 BIBLIOGRAPHY Adams, G . E . (1973) C h e m i c a l r a d i o s e n s i t i z a t i o n o f h y p o x i c c e l l s . B r i t i s h M e d i c a l B u l l e t i n , 2 9 , 4 8 . A l p e r , T . , e d . (1974) C e l l s u r v i v a l a f t e r l o w d o s e s o f r a d i a t i o n : t h e o r e t i c a l and c l i n i c a l  i m p l i c a t i o n s : p r o c e e d i n g s o f t h e s i x t h L . H . G r a y M e m o r i a l C o n f e r e n c e h e l d  a t B e d f o r d C o l l e g e , L o n d o n , 1 6 - 2 1 S e p t e m b e r , 1974 . W i l e y , New Y o r k . A l p e r , T . (1979) C e l l u l a r R a d i o b i o l o g y . C a m b r i d g e U n i v e r s i t y P r e s s . Adams, G . E . , F l o c k h a r t , I . R . , S m i t h e n , C . E . , S t r a t f o r d , I . J . , Wardman, P . , and W a t t s , M . E . ( 1 9 7 6 ) . E l e c t r o n a f f i n i c s e n s i t i z a t i o n . V I I : A c o r r e l a t i o n b e t w e e n s t r u c t u r e s , o n e - e l e c t r o n r e d u c t i o n p o t e n t i a l s , and e f f i c i e n c i e s o f n i t r o i m i d a z o l e s i n h y p o x i c c e l l r a d i o s e n s i t i z e r s . R a d i a t i o n R e s e a r c h , 6 7 , 9 . A s q u i t h , J . C . , F o s t e r , J . L . , W i l l s o n , R . L . , I n g s , R . , and M c F a d z e a n , J . A . (1974) M e t r o n i d a z o l e ( " F l a g y l " ) . A r a d i o s e n s i t i z e r o f h y p o x i c c e l l s . B r i t i s h J o u r n a l o f R a d i o l o g y , 4 7 , 4 7 4 . B a c q , Z . M . , and A l e x a n d e r , P . (1961) F u n d a m e n t a l o f R a d i o b i o l o g y . Pergamon P r e s s . B e g g , A . C . , S h e l d o n , P . W . , and F o s t e r , J . L . (1974) D e m o n s t r a t i o n o f r a d i o s e n s i t i z a t i o n o f h y p o x i c c e l l s i n s o l i d t u m o r s by m e t r o n i d a z o l e . B r i t i s h J o u r n a l o f R a d i o l o g y , 4 7 , 3 9 9 . B e n d e r , M . A . , and G o o c h , P . C . (1962) The k i n e t i c s o f X - r a y s u r v i v a l o f mammal ian c e l l s i n v i t r o . I n t e r n a t i o n a l J o u r n a l o f R a d i a t i o n B i o l o g y , 5 , 133 . B e v i n g t o n , P . R . (1969) D a t a r e d u c t i o n and e r r o r a n a l y s i s f o r t h e p h y s i c a l s c i e n c e s . M c G r a w - H i l l , I n c . , U . S . A . 96 B r a d y , L . W . , e d . (1980) R a d i a t i o n S e n s i t i z e r s ; T h e i r Use i n t h e C l i n i c a l Management o f C a n c e r . Masson P u b l i s h i n g U . S . A . , I n c o r p o r a t e d . B r o s i n g , J . W . (1983) Measurements o f C e l l S u r i v i v a l a t Low Doses o f R a d i a t i o n . PhD T h e s i s , U n i v e r s i t y o f B r i t i s h C o l u m b i a , C a n a d a . U n i v e r s i t y o f B r i t i s h C o l u m b i a , C a n a d a . C h a d w i c k , J . D . , and L e e n h o u t s , H . P . (1981) The M o l e c u l a r T h e o r y o f R a d i a t i o n B i o l o g y . S p r i n g e r - V e r l a g . Chapman, C . J . , and G i l l e s p i e , C . J . (1981) R a d i a t i o n - i n d u c e d e v e n t s and t h e i r t i m e s c a l e i n mammalian c e l l s . Advancements i n R a d i a t i o n B i o l o g y , 9_, 1 4 3 . C o l o m b o , G . , and M o r i n , G. (1963) C y t o l o g i c a l a n a l y s i s o f c o l o n i e s d e v e l o p e d f r o m mammal ian c e l l s i r r a d i a t e d i r i v i t r o w i t h X - r a y s . E x p e r i m e n t a l C e l l R e s e a r c h , 2 9 , 268 . Denekamp, J . , and H a r r i s , S . R . (1975) T e s t s o f two e l e c t r o n - a f f i n i c r a d i o s e n s i t i z e r s i n v i v o u s i n g r e g r o w t h o f an e x p e r i m e n t a l c a r c i n o m a . R a d i a t i o n R e s e a r c h , 6 1 , 1 9 1 . Denekamp, J . , M i c h a e l , B . D . , and H a r r i s , S . R . (1974) H y p o x i c c e l l r a d i o s e n s i t i z e r s : C o m p a r a t i v e t e s t s o f some e l e c t r o n a f f i n i c compounds u s i n g e p i d e r m a l c e l l s u r v i v a l _ in v i v o . R a d i a t i o n R e s e a r c h , 6 0 , 119 . D i s c h e , S . (1978) H y p o x i c c e l l s e n s i t i z e r s i n r a d i o t h e r a p y . I n t e r n a t i o n a l J o u r n a l o f R a d i a t i o n O n c o l o g y , B i o l o g y , and P h y s i c s , 4 ,^ 157 . D i s c h e , S . (1980) M i s o n i d a z o l e i n t h e c l i n i c a t Mount V e r n o n . I n : R a d i a t i o n S e n s i t i z e r s : T h e i r Use i n t h e C l i n i c a l Management o f C a n c e r . B r a d y , L . W . , e d . , Masson P u b l i s h i n g U . S . A . , I n c . 3 4 9 . 97 D u r a n d , R . E . and B r o w n , S . M . (1980) H y p o x i c c e l l s e n s i t i z e r e f f i c a c y i n m u l t i f r a c t i o n e x p o s u r e s o f an i n v i t r o tumor m o d e l . I n : R a d i a t i o n S e n s i t i z e r s : T h e i r Use i n t h e C l i n i c a l Management o f  C a n c e r . Masson P u b l i s h i n g U . S . A . , I n c . 1 5 2 . E l k i n d , M . M . , and S u t t o n , H. (1959) X - r a y damage and r e c o v e r y i n mammal ian c e l l s i n c u l t u r e . N a t u r e , 1 8 4 , 1 2 9 3 . E l k i n d , M . M . , and W h i t m o r e , G . F . (1967) The R a d i o b i o l o g y o f C u l t u r e d Mammalian C e l l s . Go rdon and B r e a c h , New Y o r k . F a d d e g o n , B . A . (1982) D o c u m e n t a t i o n o f t h e s o f t w a r e o f t h e a u t o m a t e d low dose a s s a y s y s t e m (ALDAS) . B i o p h y s i c d e p a r t m e n t , B r i t i s h C o l u m b i a C a n c e r R e s e a r c h C e n t r e , V a n c o u v e r , B r i t i s h C o l u m b i a , ( u n p u b l i s h e d ) F o s t e r , J . L . , and W i l l s o n , R . L . (1973) S e n s i t i z a t i o n o f a n o x i c c e l l s by m e t r o n i d a z o l e . B r i t i s h J o u r n a l o f R a d i o l o g y , 4 6 , 234 . F r i c h e , H . , and H a r t , E . J . (1966) C h e m i c a l D o s i m e t r y . I n : R a d i a t i o n D o s i m e t r y , vo lume I I , I n s t r u m e n t a t i o n . A t t i x , F . H . , and R o e s c h , W . C . , e d . A c a d e m i c P r e s s , New Y o r k . 126 . G r o t e , S . , J o s h i , G . , and R e v e l l , S . (1981a) A method f o r t h e s c r u t i n y o f l i v e mammalian c e l l s i n c u l t u r e and f o r t h e measurement o f t h e i r p r o l i f e r a t i v e a b i l i t y a f t e r X - i r r a d i a t i o n . I n t e r n a t i o n a l J o u r n a l o f R a d i a t i o n B i o l o g y , 3 9 ( 4 ) , 377 . G r o t e , S . , J o s h i , G . , and R e v e l l , S . (1981b) O b s e r v a t i o n o f r a d i a t i o n - i n d u c e d chromosome f r a g m e n t l o s s i n l i v e mammal ian c e l l s i n c u l t u r e , and i t s e f f e c t on c o l o n y - f o r m i n g a b i l i t y . I n t e r n a t i o n J o u r n a l o f R a d i a t i o n B i o l o g y , 3 9 ( 4 ) , 395 H a l l , E . (1973) R a d i o b i o l o g y f o r t h e R a d i o l o g i s t . H a r p e r and Row, New Y o r k . 98 H e y e , R . R . , K i e b l e r , E . W . , A r n z e n , R . J . , and T o l m a c h , L . J . (1981) M u l t i p l e x e d t i m e - l a p s e p h o t o m i c r o g r a p h y o f c u l t u r e d c e l l s . J o u r n a l o f M i c r o s c o p y , p a p e r #J30006 . I n t e r n a t i o n a l C o m m i s s i o n on R a d i a t i o n U n i t s and M e a s u r e m e n t s . (1970) R e p o r t 1 6 , L i n e a r E n e r g y T r a n s f e r . J a k o b y , W . B . , and P a s t o n , I . H . , e d . (1979) Methods i n E n z y m o l o g y , Volume L V I I I : C e l l C u l t u r e . A c a d e m i c P r e s s , New Y o r k . K e l l e r e r , A . M . , and B r e n o t , J . (1974) On t h e s t a t i s t i c a l e v a l u a t i o n o f d o s e - r e s p o n s e f u n c t i o n s . R a d i a t i o n and E n v i r o n m e n t a l B i o p h y s i c s , 1 1 , 1 . K e l l e r e r , A . M . , and R o s s i , H . H . (1978) G e n e r a l i z e d f o r m u l a t i o n o f d u a l r a d i a t i o n a c t i o n . R a d i a t i o n R e s e a r c h , 7 5 ( 3 ) , 4 7 1 . Kemper , H . , K e l l e y , S . , K a l l m a n n , R. (1980) S t a n d f o r d a u t o m a t e d c e l l / c o l o n y a u t o r a d i o g r a p h i c s c a n n e r (SACCAS) u s e r g u i d e . R a d i o b i o l o g y R e s e a r c h D i v i s i o n , D e p a r t m e n t o f R a d i o l o g y , S t a n d f o r d U n i v e r s i t y S c h o o l o f M e d i c i n e , S t a n d f o r d , C a l i f o r n i a , ( u n p u b l i s h e d ) K o r b e l i k , M . , P a l c i c , B . , and S k a r s g a r d , L . D . (1981) R a d i a t i o n - e n h a n c e d c y t o t o x i c i t y o f m i s o n i d a z o l e . R a d i a t i o n R e s e a r c h , 8 8 , 3 4 3 . M c N a l l y , N . (1976) The e f f e c t o f a change i n r a d i a t i o n q u a l i t y on t h e a b i l i t y o f e l e c t r o n a f f i n i c s e n s i t i z e r s t o s e n s i t i z e h y p o x i c c e l l s . I n t e r n a t i o n a l J o u r n a l o f R a d i a t i o n B i o l o g y , 2 9 , 1 9 1 . M o o r e , B . A . (1976) R a d i o s e n s i t i z i n g and t o x i c e f f e c t s o f R o - 0 7 - 0 5 8 2 i n h y p o x i c mammalian c e l l s . MSc . T h e s i s , U n i v e r s i t y o f B r i t i s h C o l u m b i a , C a n a d a . M o o r e , B . A . , P a l c i c , B . , and S k a r s g a r d , L . D . (1976) R a d i o s e n s i t i z i n g and t o x i c e f f e c t s o f t h e 2 - n i t r o i m i d a z o l e R o - 0 7 - 0 5 8 2 h y p o x i c mammalian c e l l s . R a d i a t i o n R e s e a r c h , 6 7 , 4 5 9 . 99 O k a d a , S . (1970) R a d i a t i o n B i o c h e m i s t r y . A c a d e m i c P r e s s , New Y o r k . P a l c i c , B . , S k a r s g a r d , L . D . , 1978 . C y t o t o x i c i t y o f m i s o n i d a z o l e and DNA damge i n h y p o x i c mammalian c e l l s . B r i t i s h J o u r n a l o f C a n c e r , 3 7 , S u p p l e m e n t I I I , 5 4 . P a l c i c , B . , B r o s i n g , J . , and S k a r s g a r d , L . D . , 1982 . S u r v i v a l measurements a t l o w d o s e s : Oxygen enhancement r a t i o . B r i t i s h J o u r n a l o f C a n c e r , 4 6 , 9 8 0 . P a r k e r , L . , S k a r s g a r d , L . D . , and E m e r s o n , P . T . (1969) S e n e s i t i z a t i o n o f a n o x i c mammal ian c e l l s t o X r a y s by t r i a c e t o n e a m i n e N - o x y l . S u r v i v a l and t o x i c i t y s t u d i e s . R a d i a t i o n R e s e a r c h , 3 8 , 4 9 3 . P u c k , T . T , and M a r c u s , P . L . (1956) A c t i o n o f X - r a y s on mammal ian c e l l s . J o u r n a l o f E x p e r i m e n t a l M e d i c i n e , 1 0 3 , 6 5 3 . S a k k a , M . , K a t s u t a , H . , and T a k a o k a , T . (1975) K i n e t i c s o f m i c r o c o l o n i e s o f c u l t u r e d m a m a l i a n c e l l s a f t e r gamma i r r a d i a t i o n . Tohoka J o u r n a l o f E x p e r i m e n t a l M e d i c i n e , 117 , 299 . S i n c l a i r , W.K. (1966) The shape o f t h e r a d i a t i o n s u r v i v a l c u r v e s o f mammal ian c e l l s c u l t u r e d i n v i t r o . I n : B i o p h y s i c a l A s p e c t s o f R a d i a t i o n Q u a l i t y . I A E A , V i e n n a , 2 1 . Thomson, J . E . , and R a u t h , A . M . (1974) A c o m p a r i s o n o f t h e e f f e c t i v e n e s s o f N F - 1 6 7 and m e t r o n i d a z o l e as h y p o x i c c e l l s e n s i t i z e r s o f KHT tumor c e l l s i n v i t r o . R a d i a t i o n R e s e a r c h , 6 0 , 4 8 9 . T h o m l i n s o n , R . H . , D i s c h e , S . , G r a y , A . J . , and E r r i n g t o n , L . M . (1976) C l i n i c a l t e s t i n g o f t h e r a d i o s e n s i t i z e r R o - 0 7 - 0 5 8 2 . I l l - Response o f t u m o u r s . C l i n i c a l R a d i o l o g y , 2 7 , 167 . 100 T h o m l i n s o n , R . H . , and G r a y , L . H . (1955) The h i s t o l o g i c a l s t r u c t u r e o f some human l u n g c a n c e r s and t h e p o s s i b l e i m p l i c a t i o n s f o r r a d i o t h e r a p y . B r i t i s h J o u r n a l o f C a n c e r , j ) , 5 3 9 . T o b i a s , C . A . , B l a k e l y , E . A . , Ngo , F . Q . H . and Y a n g , T . C . H . (1979) The R e p a i r - M i s r e p a i r m o d e l o f c e l l s u r v i v a l . I n : R a d i a t i o n B i o l o g y and C a n c e r R e s e a r c h . Meyn , R . E . , and W i t h e r s , H . R . , e d . Raven P r e s s , New Y o r k . 1 9 5 . T u r n e r , A . , A l l a l u n i s , M . , U r t a s u n , R . , P e d e r s e n , J . , and M e e k e r , B. (1980) C y t o t o x i c and r a d i o s e n s i t i z i n g e f f e c t s o f m i s o n i d a z o l e on h e m a t o p o i e s i s i n n o r m a l and t u m o r - b e a r i n g m i c e . I n t e r n a t i o n a l J o u r n a l o f R a d i a t i o n O n c o l o g y , B i o l o g y , and P h y s i c s ? ? ? , 6 , 1157 . ~ U r t u s u n , R . C . , B a n d , P . , Chapman, J . D . , R a b i n , H . R . , W i l s o n , A . F . , and F r y e r , C . G . (1977) C l i n i c a l p h a s e I s t u d y o f t h e h y p o x i c c e l l r a d i o s e n s i t i z e r R o - 0 7 - 0 5 8 2 , a 2 - n i t r o i m i d a z o l e d e r i v a t i v e . R a d i o l o g y , 1 2 2 , 8 0 1 . Wardman, P . (1977) The u s e o f n i t r o a r o m a t i c compunds as h y p o x i c c e l l r a d i o s e n s i t i z e r s . C u r r e n t t o p i c s i n R a d i a t i o n R e s e a r c h Q u a r t e r l y , 1 1 , 347 . 101 APPENDIX L i s t i n g s o f t h e ALDAS r o u t i n e s . The a u t o m a t e d l o w d o s e a s s a y (ALDAS) s o f t w a r e c o n s i s t s o f two m a j o r r o u t i n e s , LDALOC and LDACLASS, and f i v e s u b r o u t i n e s , MSCAN, ASCAN, B E E P , OUT50, and P L I N E . (See s e c t i o n 2 . 4 ) . T h i s a p p e n d i x c o n t a i n s a l i s t o f t h e v a r i a b l e s u s e d b y t h e ALDAS s o f t w a r e f o l l o w e d by l i s t i n g s o f t h e m a j o r r o u t i n e s and t h e two s u b r o u t i n e s MSCAN and ACAN. The s u b r o u t i n e s B E E P , OUT50, and PL INE i n v o l v e s i m p l e i n p u t and o u t p u t . These a r e n o t l i s t e d h e r e . V a r i a b l e s Found i n t h e M a i n R o u t i n e s and t h e S u b r o u t i n e s V a r i a b l e C (IDUM) CLASS(NLOC) CLASS( ICLASS ,NLOC) DX Type Comment I n t e g e r - number o f t i m e s c l a s s i f i c a t i o n IDUM a r r a y o c c u r s i n LDA d a t a f i l e . " - c l a s s i f i c a t i o n a t l o c a t i o n NLOC, LDALOC. " - c l a s s i f i c a t i o n a t l o c a t i o n NLOC, c l a s s i f i c a t i o n s e t I C L A S S , LDACLASS. I n t e g e r - # o f s t e p s t o be m o v e d / a c t u a l l y moved b y X m o t o r . DXH - max. # o f s t e p s t o be moved CW by X m o t o r . DXL - max. # o f s t e p s t o be moved CCW by X m o t o r . DY # o f s t e p s t o be m o v e d / a c t u a l l y moved b y Y m o t o r . DYH - max. # o f s t e p s t o be moved CW by Y m o t o r . DYL - max. # o f s t e p s t o be moved CCW b y Y m o t o r . 102 APPENDIX ( c o n t i n u e d ) V a r i a b l e FNBAK( I ) FNDAT(I ) FNLOC( I I ) FNPRN(I ) ICLASS IDUM J V A L LOC( I ,NLOC) NLOC NLOCM NSTEP NSTEPM SCAN( I ,NSTEP) SDIR XH XL XREF XSTEP YH YL YREF Type B y t e I n t e g e r I n t e g e r a r r a y I n t e g e r I n t e g e r a r r a y B y t e I n t e g e r Comment - a r r a y c o n t a i n i n g f i l e n a m e o f F o r t r a n f i l e 9 . - a r r a y c o n t a i n i n g f i l e n a m e o f F o r t r a n f i l e 7 . - a r r a y c o n t a i n i n g f i l e n a m e o f F o r t r a n f i l e 6. - a r r a y c o n t a i n i n g f i l e n a m e o f F o r t r a n f i l e 8 . - c l a s s i f i c a t i o n s e t i n LDACLASS, LDAFREQ. - m u l t i p l e u s e v a r i a b l e . - v a l u e o f b i t s 1 / P f r o m j o y s t i c k . - l o c a t i o n s 1 / P by u s e r f r o m LDALOC and u s e d b y LDACLASS. - i n d e x o f a r r a y LOC. - # o f v a r i a b l e s s t o r e d i n a r r a y LOC. - i n d e x o f a r r a y SCAN. - # o f v a r i a b l e s s t o r e d i n a r r a y SCAN. - a r r a y w i t h l o c a t i o n s 1 / P by u s e r f r o m LDAREF and u s e d by LDALOC. - s c a n d i r e c t i o n f o r d i s c r e t e p o i n t s c a n . - max. v a l u e o f XSTEP p e r m i t t e d . - m i n . v a l u e o f XSTEP p e r m i t t e d . - r e f e r e n c e p o s i t i o n , X d i r e c t i o n . - p o s i t i o n o f s t a g e a l o n g X a x i s ( s i d e - t o -s i d e ) i n # o f s t e p s . - max. v a l u e o f YSTEP p e r m i t t e d . - m i n . v a l u e o f YSTEP p e r m i t t e d . - r e f e r e n c e p o s i t i o n , Y d i r e c t i o n . 103 APPENDIX ( c o n t i n u e d ) V a r i a b l e YSTEP Type B y t e CDTNX CDTNY I END KIND NX NY SPEED XBIT YBIT XDIR YDIR XJOY YJOY I n t e g e r B y t e I n t e g e r B y t e I n t e g e r Comment - p o s i t i o n o f s t a g e a l o n g Y a x i s ( f r o n t - t o -b a c k ) i n # o f s t e p s . - b y t e o u t p u t t o p o r t 24 f o r s t e p p i n g t h e m o t o r s and p o r t 25 f o r t h e j o y s t i c k s p e a k e r s . - no X m o t o r m o t i o n i f CDTNX = 0 . - no Y m o t o r m o t i o n i f CDTNY = 0 . - # o f b e e p s 0 / P by s u b r o u t i n e B E E P . - f i x e d s c a n s p e e d i n s u b r o u t i n e ASCAN i f KIND = 1. E l s e s p e e d i s c o n t r o l l e d by u s e r t h r o u g h j o y s t i c k . - # o f s t e p s t o o u t p u t t o X m o t o r i n i n t e r n a l l o o p , s u b r o u t i n e MSCAN. - # o f s t e p s t o o u t p u t t o X m o t o r , s u b r o u t i n e ASCAN. - # o f s t e p s t o o u t p u t t o Y m o t o r i n i n t e r n a l l o o p , s u b r o u t i n e MSCAN. - # o f s t e p s t o o u t p u t t o Y m o t o r , s u b r o u t i n e ASCAN. - c o n t r o l s maximum m o t o r s p e e d t h r o u g h DO LOOP c l o c k . - 0 - no m o t i o n , 1-CW m o t i o n , 2-CCW m o t i o n , X m o t o r . - 0 - no m o t i o n , 1-CCW m o t i o n , 2-CW m o t i o n , Y m o t o r . - - 1 -CCW m o t i o n , 1-CW m o t i o n , 0 - no m o t i o n , X m o t o r . - - 1 -CCW m o t i o n , 1-CW m o t i o n , 0 - no m o t i o n , Y m o t o r . - d i g i t a l v a l u e o f X ( s i d e - t o - s i d e ) a x i s o f j o y s t i c k . - d i g i t a l v a l u e o f Y ( b a c k - t o - f r o n t ) a x i s o f j o y s t i c k . LDALOC FOR PAGE 01 cccccc CCCCCC Location scan for low dote assay. CCCCCC BYTE OPTION.KIND.SDIR.LINE( 134) , NL F NE .PAGE (AO) BYTE FNLOCdl > .FNDAT(11 ) .FNPRN(11) .CLASS(400) . PARK(7) INTEGER XSTEP.YSTEP.LOC(2.400).SCAN(2.200>.DX.DY.JVAL.XREF INTEGER YREF . DXL . t'XH, DYL .DYH. XL , XH. YL. YH. NSTEP. NSTEPM DATA FNLOC/'L'•'D'»'A'»'L'»'0'.'C'»32»32>'D'»'A'.'T'/ 1 FNDAT/32.32.32.32.32.32.32.32.'L'.'D'.'A'/ 2 FNPRN/'L' .'I>'.'A'.'P'.'R','I','N'»'T'.'0'.'U'. 1000 FORMATC/IX'HI THERE '/ 1' WELCOME TO THIS LI'A PROGRAM FOR THE RENOWNED BIOPHYS '. 2' GUYS.'/' PROGRAMMER NONE OTHER THAN B.A.FADDEGON!!'/ 3' EnJov cour.tir.sM!') 1200 FORMAT!/' LOCATION SCAN MENU. (Enter option froa c o n s o l s ) ' / 1/' A: St a r t reaular scan. '/ 2' B: St a r t d i s c r e t e point scan. ') 1201 FORMATC C! Define scan boundaries, reference p o i n t . ' . 1' and scar, points i f any.'/IX.'D! Redefine scan'. 2' parameters.'/IX*'E! Subait p r i n t o u t to p r i n t e r . ' / l X . 6'Z! E x i t routine.'/) 1202 F0RMAT(/1X' ENTER LETTER OF DESIRED OPTION! ') 1210 FORMAT(A1) 1300 FORMAT(/4X.'PARAMETER'»9X»'RANG£ VALUE COMMENT'/IX. l'A: SPEED'.lax.'0-10'.15.' Motor speed. 10 = aaxiaua.'/lX. 2'B! CELL COUNT'»9X.'1-40' . IS.' Max. no. of locations!10'. 3' to be recorded.'/IX.'CI COINCIDENCE CHECK 0-1'. 415.' 0*Autoaatic coincidence check.'/34X. 5'l»Coincidence check froa MANUAL SCAN'/34X'ALTERNATIVES. '/ 4' D! COINCIDENCE RANGE 0-127'.15.' Distance in which to ' 7/34X.'search for coincidences.'/ B' SCAN PATTERN PARAMETERS!'/' E! LINE SPACING'* 94X. '1 - 127'.15.' Nuaber of sters*10 between scan l i n e s . ' / . 1- F ! POINT SPACING' .SX.' 1-127' . 15. ' Nu.be r of stepsalO '. 2'between scan points'/34X.'for d i s c r e t e point scan.'/ 3' G! DATA FILE'.1IX.'0-9'.15.' Nuaber. n. d e f i n i n g data'. 4' f i l e for p o i n t ' / 3 4 X . ' r e t r i e v a l . 'LDALOCn.DAT'.'//) 1305 FORMAT(' ENTER LETTER OF PARAMETER TO CHANGE! '. I '(Enter 2 to e x i t ) ' ) 1320 FORMAT(' ENTER NEU VALUE FOR PARAMETER ' . A l . ' ! *) 1330 FORMAT(13) 1340 FORMAT(' PARAMETER OUT OF ALLOWED RANGE! ') 2000 FORMAT( ' RECORDED LOCATION '.14.'• ( '.16.'.'.14. 1 ') CLASSIFICATION '.ID 2010 FORMAT ( ' STEPS MOVED » ('.15.'.'.15.') LOCATION - C I S . 1 '.•.15.') NSTEP - '.14) 2020 FORMAT( ' OUTPUT DATA FILE IS * ' ,8A1. ' . ' .3A1. ' • . ' . I ' DATA IS OUTPUT TO THE P DISK.') 2030 FORMAT(' NOW READING FROM LOCATION FILE •'.SA1.'.'.3A1.'•') 3010 FORMATdX.414.215.14) 3011 F0RMAT(1X.2I4.I1.4X.2I4.I1.4X.2I4.I1.4X.2I4.I1.4X.2I4.I1.4X, 1 2I4.I1.4X) 3111 FORMATdX.2014) 4000 F0RMAT(4I4,215,14) 4001 F0RMAT(20I4) 4010 FORMAT(9A1) 4060 FORMAT(80A1) S000 FORMAT(' THIS IS THE LOCATION SCAN FOR THE LOW DOSE ASSAY.'/ 1 ' FAMILIARIZE YOURSELF UITH THESE SIGNALS!'/ 2' One beep- input from Joystick buttons.'/ LDALOC FOR PAGE 02 3 ' Two beeps- J o y s t i c k c o n t r o l s scan speed.'/ 4' Three beeps- J o y s t i c k c o n t r o l s d i r e c t i o n and speed*'/ 5' Five beeps- l a s t scan p o i n t . ' / / 4' ENTER FILENAME FOR DATA STORAGE (UP TO 8 CHARACTERS >! ') 5005 FORMAT(' USE JOYSTICK TO LOCATE REFERENCE POSITION.'./. 1 5X.'1-Ref. p o s i t i o n found. 2.3.4-Exit routine.'. 2 ' (No data output)') 5010 FORMAT(' MANUAL SCAN ALTERNATIVES! '/ 1 5X.'l-Record l o c a t i o n . 2-Record l o c a t i o n with'. 2 ' c l a s s i f i c a t i o n . '/5X» '3-Coincidence check. '»5X» 3 '4-Resuae scan.') 5020 FORMAT(' AUTOMATIC SCAN ALTERNATIVES! '/ 1 5X.'1.2-Go to Manual scan. 3-Delete l a s t recorded'. 2 ' lo c a t i o n . ' /5X *' 4-Return staae to r e f . 3 'erence p o s i t i o n and e x i t * ' ) j 5025 FORMATC DISCRETE POINT SCAN ALTERNATIVES! '/ 1 5X.'l-Record l o c a t i o n . 2-Go to Manual scan.'/ 2 5x*'3-Resuae scan. 4-Return staae to r e f . 3 'erence p o s i t i o n and e x i t . ' ) 502B FORMAT(' LOCATION'.15»' DELETED (JUST LIKE YOU WANTED! >') S030 FORMAT(' CLASSIFICATION! Enter u s i n s J o y s t i c k buttons.') 5040 FORMAT(' POSSIBLE COINCIDENCE. ALTERNATIVES!'/ 1 5Xr'1.2.3.4-Move stase to l o c a t i o n of coincidence.') 5045 FORMAT(' COINCIDENCE CHECK COMPLETED•') 5060 FORMAT ( ' HANG IN THERE TIL I GET THIS DATA OUT....') 50B0 FORMATC Movina staae to r e f . p o s i t i o n . Watch your hands*'/ O 1 ' (YOU can stop i t with the Jo y s t i c k or the buttons)') | 5100 FORMAT(' THIS IS THE REFERENCE SCAN FOR THE LOW DOSE ASSAY.'/ . 1SX'FANILIARIZE YOURSELF UITH THESE SIGNALS!'/ 25X»'0ne beep- input f r o . J o y s t i c k buttons.'/ 35X.'Two beeps- J o y s t i c k c o n t r o l s scan speed.'/ ' 45X,'Three beeps- J o y s t i c k c o n t r o l s d i r e c t i o n and speed.'/ SSX'Five beeps- a l l done s i s n s l . ' / / 6' SCAN LOCATIONS ARE STORED IN FILE •'.8A1.'.'.3A1.'•.'/) 5110 FORMAT<' LOCATE ONE BOUNDARY CORNER. OPTIONS! '/ 1 5X.'1-Location found. 2.3.4-Exit routine.') 5120 FORMAT(' LOCATE OPPOSITE BOUNDARY CORNER. OPTIONS! '/ 1 SX.'1-Location found. 2.3.4-Exit routine.') 5130 FORMAT(' LOCATE REFERENCE POSITION OPTIONS!'./. 1 SX.'l-Ref. p o s i t i o n found. 2,3.4-Exit routine.'. 2 ' (No data output)' ) S140 FORMAT(' LOCATE SCAN POINTS. OPTIONS! '/ ] 1 SX.'l-Record l o c a t i o n . 2.3-Return staae to r e f . 2 'erence p o s i t i o n and e x i t . ' / 3 5x.'4-Exit routine. (Staae not Moved.)') 4200 FORMATC LOCATION SCAN FOR FILE •',8A1, '.'.3A1.'•!'/ 1 4X»'X'»5X»'Y',3X,'CLASS'»3X.'X'»5X.'Y'»3X.'CLASS'. 2 4X» 'X' »5X, 'Y' ,3X» 'CLASS' »3X. 'X' .SX. ' Y'. 3X . 'CLASS',/) 6210 FORMATdX,1216) 6220 FORMAT(' REFERENCE POINTS STORED IN FILE •'.8A1,'.',3A1. I ' - ! ' / ' XL«'.I5.' XH""'.I5.' YL-'.IS.' YH»'.I5.' XREF='. 2 15.' YREF=' . I5./4X. 'X' ,5X, 'Y' »5X. 'X' >5X» 'Y' »5X» 'X' »5X. 3 'Y'.5X»'X'.SX.'Y'.5X.'X'.5X.'Y'.5X.'X'.5X.'Y'/) URITE(S.IOOO) 1 C Set ADC O/p ports 24 and 25. I CALL 0UT(24.15> CALL 0UT(25>25> | C Open f i l e 8 ( f o r p r i n t o u t ) . CALL 0PEN(8.FNPRN.2> ; C Set proaram parameters f o r d e f a u l t values. LDALOC FOR PAGE 03 PARM( 1 )«8 PARN(2)"40 F'ARM ( 3 >=0 r-ARrt(4)»50 PARH<5)*30 F'ARM (A ) * 10 F*ARH< 7)»0 FNLOC(7)«PARn<7>T4B C Location scan menu. 5 IDUrt-3000 URITE(S,1200) DO 6 I-l.IDUH 6 CONTINUE URITEI5.1201) DO 7 I-l.IDUM 7 CONTINUE URITE(S.1202) READ (5 r 1210) OPT ION IF(0PTI0N.EQ.45)G0T0 10 IF(0PTION.EO.44)G0T0 10 IF(0PTI0N.E0..47>G0T0 40 IF(0PTI0N.EQ.4B>G0T0 50 IF(0PTI0N.EQ.A9>G0T0 60 IF(OPTION.EO..?0)GOTO ?0 GOTO 5 C Open f i l e 7 ( f o r recorded l o c a t i o n s ) . 10 «RITE(5.5000> READ(5»4010)(FNDAT(I), I*1»B> DO 105 IM.S 105 IF(FNDAT(I).EQ.13)FNDAT(I>=32 URITE(5.202OHFNDAT(I > .1-1,11) CALL 0PEN(7.FNDAT»2) C Find reference p o s i t i o n . URITE(5.5005) DX=0 DY-0 JVAL«1S CALL BEEP(3> CALL MSCAN(OX. D Y i JUAL . -20000 . 20000. -20000.20000.PARN (1)) IF(JUAL.NE.14)G0T0 5 C I n i t i a l i z e program v a r i a b l e s . NSTEP'l NL0C*0 SDIR.O DO 130 I'1.400 130 CLASS(I)=0 KIND«1 IF(0PTI0N.E0.«3)KIND«0 C Read l o c a t i o n s of scan reference points and automated scan poi C fro» f i l e LDALOC.DAT. CALL OPEN (6 . FNLOC,1) URITE(5.2030)(FNL0C(I> >I»Ir11 ) READ(6,4000 > XL.YL.XH.YH.XREF.YREF,NSTEPM 1F(0PTI0N.E0.44>G0T0 20 IF(NSTEPM.E0.0)G0T0 20 READ(6, 4001 >(SCAN(1.1).SCAN(2,1).I"l.NSTEPM) GOTO 107 C. Generation of scan points for resuler and d i s c r e t e point scans 20 IDUrl«*F-ARrl(5)»I0 NSTEF'M=0 LDALOC FOR PAGE 04 DO 210 I>lt200.2 SCAN<l.I>-XLtIDUMt((Itl)/2> SCAN!1.Itl I'SCANU .I> NSTEPM-NSTEPHT2 210 I F ( S C A N ( 1 . I ) T I D U M . G T . X H ) G 0 T 0 220 220 IDUM*YLtPARM(a>»10 JDUM»YH-PARM(4>«10 DO 230 I»l.NSTEPM.2 SCAN(2.I)-IDUH SCAN(2.1*1l-JOUM IDUrfJBUM 1 230 JDUM-SCAN(2,I > ! 107 ENDFILE 6 ! XSTEP«XREF I YSTEP-YREF C Auto scan, prosrem chooses d i r e c t i o n from the array SCAN. User C c o n t r o l s speed of scan with J o y s t i c k . C JUAL returned f r o * ASCAN provides A l t e r n a t i v e s 1. 113 IF(NL0C.GE.PARM(2)«10)G0TO 300 I IF<0PTI0N.EQ.°5)URITE<5.5020> | CALL BEEP<2> 100 IF(NSTEP. LE. NSTEF'M >D0TO 110 120 IF(NL0C.GE.PARM(2)*10)GOTO 300 — ' URITE(S.SOIO) O CALL PEEP(S) |V-T1 GOTO 250 I 110 IF(0PTI0N.EQ.45>G0T0 111 , C Determination o f next point to scan to f a r d i s c r e t e point scan. IF<SDIR.NE.0)G0T0 113 ! SDIR'l XSTEPl'SCAN(l.l) YSTEPl-SCAN(l.l) GOTO 111 113 IDUM»YSTEP1TPARM(6)*10*SDIR IF<IDUM.GE•YH.OR.IDUM.LE.YL1G0T0 117 DX-XSTEP1-XSTEP YSTEP1-IDUM DY«YSTEP1-YSTEP GOTO 11? 117 NSTEP"NSTEP+2 IF<NSTEP.GT.NSTEPri>GOT0 lOO I XSTEPl-SCANU.NSTEP) I YSTEP1-SCAN<2.NSTEP> SDIR— ItSDIR C ASCAN. 111 DX=SCAN(1,NSTEP>-XSTEP DY»SCAN< 2.NSTEP) -YSTEP 119 CALL ASCAN(DX.DY.JUAL.KIND.PARM(l)) XSTEP-XSTEPtDX YSTEP"YSTEP*DY URITEiS.20101DX.DY.XSTEP.YSTEP,NSTEP IF(0PTI0N.E0.45)G0T0 121 C Input J U A L and branch on J U A L . d i s c r e t e point scan options. I URITE(3,S025> ] CALL B E E P ( l ) 122 JUAL=INP<24> i J U A L " J U A L - ( J U A L / l o > « 1 4 I F ( J U A L . L T . 0 ) J U A L - J V A L + 1 6 IF(JUAL.E0.15)G0T0 122 DO 12? 1=1.10000 LDALOC FOR PAGE OS 129 CONTINUE IF(JVAL.EQ.I3>G0TO 200 IF(JUAL.EQ.11)G0T0 100 IF < JVAL.E0.7)GOTO 300 NLOC-NLOC+l LOC(1.NLOC)-XSTEP L0C(2.NL0C)=YSTEP CLASS(NLOC)=5 GOTO 180 C Branch on JUAL» regular scan options. 121 IF(JUAL.E0.13>NSTEP-NSTEP+1 IF(JVAL.E0.15)G0T0 100 IF(JUAL.EO.7)GOTO 300 IF(JUAL.NE.11)GOTO 200 IF(NL0C.LT.1)G0T0 113 URITE<3.5028)NL0C NLOC-NLOC-1 GOTO 113 C Manual scan. Control of speed and d i r e c t i o n u i t h J o y s t i c k . C JUAL returned froe, MSCAN provides A l t e r n a t i v e s 2. 200 URITE(5.5010> CALL BEEP<3) 230 DXL-XL-XSTEP DXH-XH-XSTEP DYL-YL-YSTEP DYH-YH-YSTEP DX-0 • DY-0 JUAL-15 CALL MSCAN < DX. DY, JUAL, DXL, DXH, DYL , DYH, PARM (1) ) XSTEP-XSTEPtDX YSTEP*YSTEP+DY URITE(3,2010)DX,DY,XSTEP,YSTEP,NSTEP C Coincidence check. IF(PARM<3>.EO.l.AND.JUAL.NE.111G0T0 193 IF(JUAL.EO.7->G0T0 193 IF(NLOC.LT. DGOTO 198 IDUM=0 DO 197 I-l.NLOC IF(L0C(1.I)+PARM<4).LT.XSTEP>G0T0 197 IF(LOC< 1,1 > -PARM (4).GT.XSTEP)G0T0 197 IF<L0C(2,I>+PARM(4).LT,YSTEP)GOTO 197 IF(LOC(2,I>-PARM(4>.GT.YSTEP1G0T0 197 IOUM-1 CALL BEEF' ( 4 > URITE(5,5040) 199 JDUM=INP(24) JDUM=JDUM-( JDUM/16)*16 IF < JDUM.LT.0)JDUM-JDUM +14 IF(JDUM.EQ.15)G0T0 199 DX«L0C<1 r I >-XSTEP DY»L0C(2.I)-YSTEP CALL BEEPI2) CALL ASCAN<DX,DY,JDUM,1,PARM(1)) XSTEP-XSTEP+DX YSTEP-YSTEP+DY URITE(5.2010JDX.DY,XSTEP,YSTEP,NSTEP 197 CONTINUE 198 URITE(5,5043> IF (F'ARM (3 > .EQ. 1 . OR. JUAL . EO .'11 > GOTO 200 LDALOC FOR PAGE 06 IF(IDUM.EO.1)GOTO 200 C Record stase l o c a t i o n . 195 IF ( NSTEP .GT. NSTEF'M. AND • JUAL. EO. 7 )GOTO 300 IF(JUAL.EO.7)GOTO 115 NLOC-HLOC+l L0C(1,NL0C)=XSTEP LOC(2.NL0C)»YSTEP CLASS(NLOC) -5 IFCJUAL.EO.14)G0T0 180 C Pause before beep. DO 500 1-1,1000 500 CONTINUE URITE(3,3030> CALL DEEP(l) C C l a s s i f i c a t i o n at t h i s staae l o c a t i o n . 400 JUAL"INP(24> JUAL-JUAL-(JUAL/16X16 IF(JVAL.LT.O)JUAL-JUAL+16 IF(JUAL.E0.15)G0T0 400 NDUM-0 IFCJUAL.EO.141NDUM-1 IF< JUAL.EO. 131NDUM-2 IF(JUAL.E0.111NDUM-3 IF( JUAL. EO. 7 >NDUM-4 CLASS(NL0C1-NDUM 180 URITE (5, 2000) NLOC, LOC < 1, NLOC ) ,LOC( 2, NLOC), CLASS (NLOC ) IF(NSTEP.GT.NSTEPM)GOTO 120 GOTO 115 C Output data to data f i l e 7 and p r i n t o u t to f i l e 8. 300 IF(NL0C.E0.0)G0TO 600 WRITE(3.5060> URITE(7.3010)XL,YL r XH,YH,XREF.YREF,NLOC WRITE(7,3Oll)<L0C(l,I),LOC<2,I),CLASS(I),I-l,NL0C) ENDFILE 7 URITE(8,6200 HFNDAT(I),1-1,11) URITE(6.6210)((LOC<J.I>,J-l,2),CLASS(I>,1-1,NLOC) URITE<5,2020)<FNDAT(I),1-1,11) C Return stade to reference p o s i t i o n . 600 URITE(5.S0B0> DX-XREF-XSTEP DY-YREF-YSTEP CALL BEEPC2) CALL ASCAN(DX,DY»JVAL,1»PARM(1>) GOTO 3 CCCCCC CCCCCC Reference and scan point l o c a t i o n scan f o r low dose assay. CCCCCC C Open f i l e 6 ( f o r data output). 40 CALL 0PEN(6,FNL0C,1> C One boundary corner l o c a t e d . URITE(5,5100)(FNLOC(I),1=1,11) WRITE(5,5110) CALL BEEP(3) CALL MSCAN(DX,DY,JUAL,-20000,20000,-20000,20000.PARM(1)) IF(JUAL.NE.14)G0T0 5 C Opposite boundary cornver l o c a t e d . URITE(S.5120> CALL BEEP(3) CALL MSCAN(DX.DY.JUAL.-20000.20000.-20000.20000.PARM(1)) IF(JVAL.NE.141G0T0 5 107 LDACLASSFOR PAGE 01 ccccc CCCCCC C l a s s i f i c a t i o n scan for low dose assay. CCCCCC BYTE FNDAT(11),FNPRN<11)•OPT ION.PARM(6) .PARM4 .CLASS(5.400) BYTE LINE(134).NLINE.PAGE<60> INTEGER XSTEP.YSTEP.LOC(2.400).DXtDYtJVAL.XREF.YREF. 1 DXL.DXH.DYL.DYH.XL.XH.YL.YH.C(6) DATA DUMI.DUM2.DUM3.DUN4/1.0.2.0.3.0.4.0/ DATA FNDAT/32.32.32.32.32.32.32.32,'L'.'D'.'A'/ 1 FNPRN/'L' .'D'.'A'.'P'.'R'.'I'.'N'.'T'.'O'.'U'.'T' 1200 FORMAT(/' CLASSIFICATION SCAN MENU. (Enter option froa c', 1 'onsole)'//' A! Start scan.') 1201 FORMAT( ' C: C a l c u l a t e freouency of c l a s s i f i c a t i o n . ' / 1' D: Redefine scan paraaeters.') 1202 FORMATC E.' Subniit p r i n t o u t to p r i n t e r ' / 1 ' Z: E x i t r o u t i n e . ' / / ' ENTER LETTER OF DESIRED OPTION: 1210 FORMAT(Al) 1300 F0RMAT(/4X.'PARAMETER'.11X.'RANGE VALUE COMMENT'/IX. l'A: SPEED'.17X.'0-10'.16.' Motor speed. 10=«jxiaua.'/IX. 2'B: PRINTOUT',14X.'0-2'.17.' 0»No Printout.'/3SX,'1-Class'. 3 ' i f i c a t i o n freouencies only. '/38X . ' 2=A11 printout.'/IX. 4'C: SET TO CLASSIFY'.7X.'0-6'.17.' 0-No c l a s s i f i c a t i o n . ' / S3SX. ' 1-5-Set to c l e s s i f y.'/38X >'6"Fi r s t set with no c l a s s i ' , 4 ' f i c a t i o n s . ' / ' COLONY LOCATION CONSTRAINT PARAMETERS!'/IX. 7'D! CLASSIFICATION SET 0-5'.17.' 0«No constraint.'/38X. 8'l-5«Set containina c o n s t r a i n t . ' / ' E! CLASSIFICATION '. 9'VALUE 0-5'.17.' Class of l o c a t i o n s to be scanned.'/ 11X.' CLASSIFICATION FREQUENCY CONSTRAINT PARAMETERS!'/ 21X.'F! CLASSIFICATION SET 0-5'.17.' 0«No Constra'. 3'int.'/38X.'l-5«Set containina c o n s t r a i n t . ' / / ) 1305 FORMATC ENTER LETTER OF PARAMETER TO CHANGE! '. 1 '(Enter Z to e x i t ) ') 1320 FORMATC ENTER NEU VALUE FOR PARAMETER ' . A l . ' ! ') 1330 FORMAT(12) 1340 FORMATC PARAMETER OUT OF ALLOWED RANGE! ') 2000 FORMAT ( ' "STEPS MOVED = C . 15, ' .'. 15. ' ) LOCATION =• ('.15. 1 '.'.15.') NLOC - '.14) 2010 FORMATC RECORDED LOCATION '.I4,'« ('.14.'.'.14. 1 ') CLASSIFICATION '.ID 2020 FORMATC OUTPUT DATA FILE IS •'.8A1.'.'.3A1. ' • . '. 1 ' DATA IS OUTPUT TO THE B DISK.') 2030 FORMATC NOW READING FROM LOCATION FILE •'.8A1.'.',3A1.'•') 2040 FORMATC THIS IS YOUR'.12.'TH CLASSIFICATION.') 2100 FORMAT(' UNEXPECTED CLASSIFICATION! CLASS('.11.'.'.13. 1 '> •'.16.'. ' > 2110 FORMAT(/' LDA FREOUENCY OF CLASSIFICATION RESULTS FOR FIL'. 1 'E •'>8A1.'.'.3A1.'•.'/' SET '.'CLASS 0',3X,'CLASS 1', 2 3X,'CLASS 2'r3X.'CLASS 3'.3X.'CLASS 4'»3X»'CLASS 5'/) 2115 FORMAT(' CLASSIFICATION SET'.13.' CONSTRAINED TO CLA'. 1 'SSIFICATION',13.'. RESULTS FOLLOW!') 2120 FORMAT(3X.11.16.4X.16.4X.16.4X.16.4X.16.4X.16> 3000 F0RMATI4I4.2I5.I4) 3001 FORMAT(214,511.214.511.214.511.214.511.214.511.214,SID 3002 FORMAT <?X. 511.8X.511 . 8X. 511. 8X. 511. 8X . 511. 8X. 511) 3010 FORMATdX.414.215.14) 3011 FORMAT(IX.214.511.214.SI1.214.SI 1.214.SI 1.214.511.214.511) 3100 F0RMAT(26X.I4) 4010 FORMAT(8A1) 4060 FORMAT(BOA 1) 5 0 0 0 FORMATC THIS IS THE CLASSIFICATION SCAN FOR THE LOU'. LDACLASSFOR PAGE 02 1 ' DOSE ASSAY.'/ 2 ' FAMILIARIZE YOURSELF UITH THESE SIGNALS'. ' / 3 ' One beep- input f r o a j o y s t i c k buttons.'/ 4 ' Two beeps- J o y s t i c k controls scan speed.'/ 5 ' Three beeps- J o y s t i c k c o n t r o l s d i r e c t i o n and speed 6 ' F i v e beeps- a l l done s i g n a l . ' / / 7 ' ENTER FILENAME OF LOCATION DATA (UP TO 8 CHARACTERS)! ' 5005 FORMAT(' USE JOYSTICK TO LOCATE REFERENCE POSITION.',/. 1 /SX.'JOYSTICK OPTIONS!'/ 2 5X.'1-Ref. p o s i t i o n found. 2.3.4-Exit routine.'. 3 ' (No data output)') 5010 FORMATC MANUAL SCAN ALTERNATIVES! '/ 1 SX.'l-CIassify.'13X.'2-Reset scan coordinates.'/ 2 SX.'3,4-Resuae scan.') 5020 FORMATC Use J o y s t i c k to c o n t r o l scan treed to next '. 1 'location.'/5X.'Press any button to stop scan.') 5030 FORMATC CLASSIFICATION! Enter usina J o y s t i c k buttons.') S04O FORMAT(' CLASSIFICATION SCAN ALTERNATIVES.'/5x« 1 '1-Clessify.'13x.'2-Move '. 2 'staae.'/5X.'3-Resuae Scan.'.1 Ox.'4-Exit routine.') SOSO FORMAT(' YOU HAVE ALREADY MADE FIVE CLASSIFICATIONS '. 1 'KEENER!'/' SORRY THAT IS YOUR LIMIT.') 5060 FORMATC GOOD GOING! ANOTHER ONE DONE!!!!'/ 1 ' JUST HANG ON A SEC WHILE I SPIT OUT THE DATA.') 5080 FORMAT(' Movina staae to r e f . p o s i t i o n * Watch your hands!'/ 1' (YOU can stop i t with the Jo y s t i c k or any of the buttons)') 5100 FORMAT(' THIS IS THE CLASSIFICATION FREQUENCY PROGRAM OF'. 1 ' THE LOU DOSE ASSAY.'/ 2 ' This routine c a l c u l a t e s the nuaber of t i a e s each c l a ' * 3 ' s s i f i c a t i o n occured '/ 4 ' f o r each set of c l a s s i f i c a t i o n s i n wour LDA data f i ' * 5 ' l e . ' / / ' ENTER THE FILENAME OF YOUR DATA FILE (UP TO 8'. 6 ' CHARACTERS): ') 5110 FORMAT(/' Hope you enjoyed t h i s LDA proaraa. Good luck w'. 1 ' i t h your r e s u l t s . ' / ' FILES OUTPUT FOR YOUR VIEWING PL'. 2 'EASURE:'/' LDAPRINT.OUT- P r i n t o u t f o r t h i s session.') 6200 FORMAT(' CLASSIFICATION SCAN FOR FILE •'.BA1.'.'.3A1.'•'. 1 '. CLASSIFICATION SET '.11/ 2 4X.'X'.5X»'Y'»3X»'CLASS'.3X.'X'.5X.'Y'.3X.'CLASS'. 3 3X»'X'* SX.'Y'»3X»'CLASS'»3X»'X'»5X» 'Y'»3X.'CLASS'./) 6210 FORMAT (IX.216,IX.511*216. tX.SIl.216. IX. 511.216. IX. 511) Set ADC 0/P ports 24 and 25. CALL 0UT(24.15) CALL 0UT(25.25) Open f i l e 8 ( f o r p r i n t o u t ) . CALL 0PEN(8.FNPRN.2) Set proaraa paraaeters f o r d e f a u l t values. PARM(1)=8 PARM(2)=1 PARM(3)«6 PARM(4>-0 PARM4»PARM(4) PARM(5)-0 PARM(6)*0 C l a s s i f i c a t i o n scan aenu. 5 IDUM-.3000 WRITE(5.1200) DO 6 1=1.IDUM 6 CONTINUE WRITE!5.120D LDACLASSFOR PAGE 03 DO 7 I » 1 .1DUN 7 CONTINUE URITE(5.1202) READ(5.12I0> OPTION IF(0PTI0N.E0.65)G0T0 10 IF(0PTI0N.EQ.68)G0T0 20 IF(OPT ION.EO.47(GOTO 30 IF(0PTI0N.E0.69)G0T0 40 IF(OPTION.E0.9O)GOT0 90 GOTO 5 C Open f i l e s 7 (for recorded locations) 9 (for c l a s s i f i c a t i o n s ) . 10 URITE(5.5000> READ(5.4010)(FNDAT(I).I-1.8) DO 105 1-1.8 105 IF(FNDAT(I).EO.131FNDAT(I)-32 URI TE( 5.2020) (FNDAT ( D . I - l r l l ) CALL OPEN(7.FNDAT.2> C Find reference p o s i t i o n . URITE(5.5005) DX-0 DY-0 JUAL-15 CALL BEEPI3) CALL MSCAN(DX.DY.JVAL.-11000.11000. -11000.11000 . PARM (1) ) IF(JVAL.NE.14>G0T0 5 NLOC-0 C Read l o c a t i o n s of scan reference P o i n t s and automated scan C p o i n t s from f i l e 7. WRITE!5.2030)(FNDAT(I).1-1.11) REAIK 7.3000)XL.YL.XH.YH.XREF,YREF.NLOCM XSTEP-XREF YSTEP-YREF READ(7.3001)(L0C(1.1).LOC(2.I).(CLASS(J.I).J-l.5).I-1.NLOCM) IF(PARM(3>.EQ.6)G0T0 710 ICLASS-PARM(3) GOTO 740 710 DO 700 ICLASS-l.S DO 750 J-l.NLOCM 750 IF(CLASS(ICLASS.J).NE•0)GOTO 700 GOTO 740 700 CONTINUE URITE(S.S050) GOTO 780 760 URITE(5.2040)ICLASS C Auto scan. Program chooses d i r e c t i o n from the array SCAN. User C controls speed of scan with J o y s t i c k . JVAL unchanged by ASCAN. 50 NLOC-NLOCtl IF(NL0C.GT.NL0CM)G0T0 300 IF(PARM4.EG.0)G0T0 120 DO 110 I-NLOC.NLOCM 110 IF(CLASS(PARM4. I) .EQ.PARM(S) ) GOTO 115 GOTO 300 115 NLOC-I 120 URITE(5.3020> CALL B£EP(2> DX-LOC(1.NLOC)-XSTEP DY"L0C(2.NL0C)-YSTEP CALL ASCANIDX.DY.JUAL.1.PARM(1) ) 150 XSTEP-XSTEP+DX YSTEP-YSTEPtDY LDACLASSFOR PAGE 04 URITE(3.2000)DX.DY.XSTEP.YSTEP.NLOC C A l t e r n a t i v e s 1 from JUAL. 180 WRITE(3.5040) CALL BEEP(l) 200 JUAL-INP(24) JUAL-JUAL-(JUAL/16)*16 IF(JVAL.LT.O)JVAL-JVAL+14 IF(JUAL.E0.15)G0T0 200 IF( JVAL.EO. 131G0T0 900 IF(JVAL.E(1.7>G0TO 300 IF(PARM(3).EQ.OIGOTO SO IF<JVAL.EO.14)GOTO 600 CLASS! ICLASS.NLOO-5 GOTO 50 C Manual scan. Control of speed and d i r e c t i o n with Joystick. C JVAL O/P from MSCAN provides A l t e r n a t i v e s 2. 900 DXL-XL-XSTEP DXM-XH-XSTEP DYL-YL-YSTEP DYH—YH-YSTEP URITEIS.SOIO) DX-0 DY-0 JVAL-13 CALL BEEP(3> CALL MSCAN( DX. PY. JUAL . DXL . DXH. DYL . DYH r PARM (1 ) ) IF(JVAL.E0.131G0TO 180 XSTEP-XSTEP+DX YSTEP-YSTEP+DY URITE(3.2000)DX.DY.XSTEP.YSTEP.NLOC IF(PARN(3>.EO.0)GOTO 50 IF(JUAL.EQ. 141G0T0 600 CLASS( ICLASS.NLOO-5 GOTO 50 C Pause before beep. 600 DO 500 1-1.10000 500 CONTINUE CALL BEEP(l) C C l a s s i f i c a t i o n at t h i s stage l o c a t i o n . URITE(5.3030) 400 JVAL»INP<24) JVAL«JUAL-( JVAL/16X16 IF (JVAL . LT. 0) JVAL-JVAL+16 IF(JVAL.E0.15)G0T0 400 NDUH-0 IF(JVAL.EO.14>NDUM-1 IF(JVAL.E0.131NDUM-2 IF(JVAL.EO.11INOUN-3 IF<JVAL.EO.71NDUM-4 CLASS! ICLASS.NLOO-NDUM URITE(3.2010)NLOC.(L0C(J.NLOC),J-1.2).CLASS(ICLASS.NLOC) GOTO 50 300 CALL BEEP(5> IF(NL0CM.E0.0)GOT0 S • IF(PARM(3).EO.OIGOTO 320 URITE(5.5060> REWIND 7 URITE(7.3010)XL.XH,YL.YH.XREF.YREF.NLOCM URITE(7.3011)((LOC(J.I).J-l.2).(CLASS!K.I).K-1.5>.I-1.NLOCM> IF(PARM(2).NE.2)GOTO 310 LDACLASSFOR RAGE OS WRITE(8,6200)(FNDAT(I).I-l.11),ICLASS URITE<8,6210)<(LOC(J.I> »J-1.2> > <CLASS(K,I>,K-1,S>,I-l,NLOCH) 310 WRITE(5>2020>(FNDAT(I),1-1,11) C Return stss~e to reference p o s i t i o n . 320 WRITEC5.5080) DX-XREF-XSTEP OY-YREF-YSTEP CALL BEEP(2) CALL ASCAN(DX,DY,JUAL,1.PARK(1)> 780 ENDFILE 7 GOTO S CCCCC Parameter l i s t and d e f i n i t i o n . CCCCC 20 URITE(3>1300KPARM(I>,I>1,6> 230 URITE(5rl303) READ(5 r1210)OPT ION IF(DPTI0N.E0.?O)G0T0 S IDUM-OPTION-64 IFUDUH.LT. l.OR. I DUN. GT. 6) GOTO 20 220 URITE(5rl32O)OPTI0N R£AD(S.1330)PARndDUM> PARM4-PARM(4) JDUM-0 IF(PARM(1).LT.O.OR.PARN(1).GT.10)JDUM-1 IF(PARMC2).LT.0.0R.PARN(2).GT.2)JDUrt-2 IF(PARM(3).LT.0.0R.PARMI3).GT.6)JDUM-3 IF(PARN(4>.LT.0.0R.PARM(4).0T.5)JDUM=4 IF(PARM(5) .LT.O.OR.PARM(S) .GT.5)JDUM»5 IF(PARM(6).LT.0.0R.PARM<6).GT.S)JDUM-6 IF(JDUM.EO.0)GOT0 230 IDUM-JDUM OFTI0N=IDUM»64 URITE(5>1340> GOTO 220 CCCCC Freouency of c l a s s i f i c a t i o n s c a l c u l a t i o n for low dose assay. CCCCC C Open f i l e s 7 (for c l a s s i f i c a t i o n s ) and 8 (for p r i n t o u t ) . 30 URITE(3,3100) READ.5.4010)(FNDAT(I).I-1.8) 00 800 1-1.8 800 IF(FNDAT<I).EQ.13)FNDAT(I)=32 CALL 0PEN(7,FNDAT,2) C Read c l a s s i f i c a t i o n s . READ<7r310O)NL0CM READ(7,3002>((CLASS<IPJ>.I-l»3).J"1.NLOCM) C Loop to ca l c u l a t e c l a s s i f i c a t i o n freauencies. WRITE. 3F 21 lOXFNDAT'I). I - l r 11) IF(PARH(2>.NE.O)WRITE(8.2110>(FNDAT(I).1=1,11) JDUM»PARM(6> IF<JDUM.E0.O)GOTO 808 IC-0 802 DO 803 NL0C-1,NLOCM 803 IF(CLASS(JDUM>NL0C) .EQ.IOG0T0 801 GOTO 880 801 IF(PARM( 2) . NE. 0) URITE( 8r 21 IS) JDUM, IC WRITE(5.2US>JDUM,IC 808 DO 810 ICLASS-1.5 DO 807 NLOC-1,NLOCM 807 IF(CLASS<ICLASS.NLOC).NE.01G0TO 804 GOTO 810 806 DO BSO 1-1,6 B30 C<I)-0 DO 820 NLOC-1,NLOCM IF<JDUM.NE.O.AND.CLASS<JDUM,NLOC).NE.ICI GOTO 820 IDUM-CLASS(ICLASS,NLOC)+1 IF(IDUM,GT.6.OR.IDUM.LT.DGOTO 830 C(IDUM>-C(IDUM)tl GOTO 820 830 I DUM-1 DUM-1 WRITE(3,2100)ICLASS,NLOCIDUM IF<PARM(2).E0.O)G0TO 820 URITE(8 r 2100) ICLASS, NLOC, IDUM 820 CONTINUE WRITE(3,2120)ICLASS,(C(I),1-1,6) IF(PARM<2).EQ.0)G0T0 810 URITE<8,2120)ICLASS.(C(I).1=1,6) 810 CONTINUE 880 IC-IC+1 IF<JDUM.NE.O.AND.IC.LT.6>00TO 802 ENDFILE 7 GOTO S C C Routine to subisit f i l e BSLDAPRINT.OUT to l i n e p r i n t -C 60 REWIND B CALL 0UT50I27) CALL 0UT50(32> CALL 0UT50(32) NLINE-3 DO 663 I-l,S3 663 PAGE(I)-32 PAGE<S4)-80 PAGE<53)-63 PAGE(36)-71 PAGE(37)-6? PAGE(58)-32 PAGE(59>-48 PAGE(60>-4? DO 623 I - 1,11 423 PAGE<I) - FNPRN(I) CALL OUTSO(IO) CALL PLINE(PAGE,60) CALL OUTSO(IO) 665 READ(8,4060.END-673) (LINE(I),1=1,80) IF(NLINE.LT.63)G0T0 670 NLINE-3 DO 660 1-1,4 660 CALL OUTSOdO) PAGE(60>-PAGE(40>+1 IF(PAGE(60>.LT.S8>G0T0 667 PAGE<67)-48 PAGE(39)-PAGE(39>tl IF(PAGE(59) .LT.38)G0T0 647 PAGE(59)-4B 667 CALL PLINE(PAGE.40> CALL OUTSOdO) 670 NLINE-NLINEd CALL PLINE(LINE,80) GO TO 663 673 DO 680 I-NLINE,65 680 CALL 0UT30(10) REWIND 8 00 TO 3 C Set ADC 0/P ports 24 and 23. 90 CALL 0UT(24.1S) CALL OUT(23.0> C Al1 done 'signal. CALL BEEP(5) U R T T F . =. . =. 1 m l JT£>t» EMO LDASUB FOR PAGE 01 CCCCCC AUTOMATIC SCAN. I/P * OF PULSES TO SUP..OX.DV. CCCCCC Interrupt scan anytime with Joystick buttons. SUEtROUTI NE ASCAN< DX, DT . JVAL . KIND. SPEED ) INTEGER DX.DY.NX.NY.JVAL »YJOY BYTE KIND.SPEED.XBIT.YBIT.CDTNX.CDTNY.B NX-0 NY-0 CDTNX=1 CDTNY=1 JUAL-15 I.' Set XBIT. YBIT. XBIT-1 IF(DX.LT.0)XBIT=2 IF(DX.EO.O)XBIT-0 YBIT-2 IF(DY.LT.0)Y8IT-1 IF(IiY.EO.O>YBIT-0 C Set CDTNX. CDTNY. Output pulses to motors when CDTN-l. 100 IF(NX.GE. lABS(DX) >CDTNX=0 IF(NY.GE.IABS(DY))CDTNV-0 IF(CDTNXTCDTNY.EQ.OIGOTO 200 B=XBIT*Ct'TNX + YBIT*CDTNY*4 B-15-B CALL 0UT(24.B> CALL 0UT(24.15> NX-NX+CDTNX NY-NY+COTNY C YJOY DEFINES PULSE RATE. 400 YJ0Y-INPC26) C JUAL TO INTERRUPT. JVAL IS INPUT FROM JOYSTICK BUTTONS. JVAL=INP(24) JVAL"JVAL-(JVAL/16>*14 IF(JVAL.NE.15.AND.JUAL.NE.-1)GOTO 200 IF(YJ0Y.LT.-100)G0T0 400 C CLOCKS. IF(YJOY.GT.100)YJOY-100 IF(KIND.EO.1rYJOY-100 IEND—32-YJ0Y+3201 DO 300 I-l.IEND 300 CONTINUE IEND=301-5PEED*30 DO 500 1=1.IEND 500 CONTINUE GOTO 100 200 IF(JVAL.LT.0)JVAL-JVALrl6 IF(DX.NE.0)DX'(DX/IABS(DX1)»NX IF(DY.NE.O>DY=(DY/IABS(DY))«NY RETURN END CCCCCC THIS PROGRAM CONVERTS I/P FROM JOYSTICK TO MOTOR STEPS. CCCCCC X AND Y MOTOR SPEEDS ARE CONTROLLED INDEPENDENTLY WITH JOYSTI SUBROUTINE MSCAN(DX.DY.JVAL.DXL.DXH.DYL.DYH.SPEED) BYTE B.CDTNX.CDTNY.XBIT.YBIT.SPEED.XDIR.YDIR INTEGER JUAL.XJOY.YJOY.DX.DY INTEGER DXL.DXH.DYL.DYH XJOY=0 YJOY-0 DX-0 HY-0 NX=0 LDASUB FOR PAGE 02 NY"0 XDIR"0 YDIR-0 IEND"301-30«SPEED C INPUT FROM JOYSTICK. CALCULATE NX.NY FROM XJOY.YJOY. 120 XJ0Y=INP(23) YJ0V-INP(26> NX»2«»((XJ0Y«XJ0Y>/1900-1> IF(NX.GT.64)NX»64 NV-2»»{(YJOYtYJOY1/1900-1> IF<NY.GT.64)NY-64 C I/P FROM JOYSTICK BUTTONS TO RETURN FROM SUBROUTINE. JVAL-INPC24) JUAL-JVAL-(JVAL/14 )* 14 IF( JVAL. LT.O) JVAL-JVALM6 IF(JVAL.NE.15)G0T0 1B0 IF(NXrNY.EO.0)GOTO 120 C MOTOR DIRECTION DETERMINED. XDIR-0 IF(IABS(XJOY>.GT.431X01R-XJOY/1ABS(XJOY) YDIR-0 IF(IABS(YJ0Y).GT.43)YDIR-YJ0Y/IABS(YJ0Y) . XBIT-XDIR I F ( X B I T . L T . 0 ) X B I T - 2 YBIT-0 IF(YDIR.LT.0)YBIT-1 IF(YDIR.GT.0)YBIT-2 C LOOP. 0/P 0.1.2.4.fl.16.32.64 pulses to motors. (Depends on C NX.NY). DO 170 1-1.64 CDTNX-0 CDTNY-0 C CDTN SET. THIS CONTROLS MOTOR SPEED. NO MOTION UNLESS CDTN-IF(((I*NX)/64)«44.EO.I»NX)CDTNX-1 IF<((I«NY)/64)«64.E0.I*NY)CDTNY=1 C CHECK FOR STAGE OVERRUN• 1F(DX.OT.DXL)GOTO 130 IF(XDIR.E0.-11CDTNX-0 130 IF(DX.LT.DXH)GOTO 140 IF(XDIR,Ea.l)CDTNX-0 140 IF(DY.GT.DYL>GOTO 150 IF(YDIR.EO.-l)CDTNY-0 130 IF(DY.LT.DYH)G0TO 160 IF(YDIR.E0.1)CDTNY-0 C SET OUPUT BYTE TO ADC ( U H I C H OUTPUTS TO MOTOR TRANSLATORS). 140 B»XBIT»CBTNX+YBIT»CDTNY*4 B-15-B CALL 0UTC24.B) CALL 0UT(24.13) DX-DXtXDIR*CDTNX C Clock to c o n t r o l ranse of speeds. DO 190 J-l.JEND 190 CONTINUE 170 HY-DYTYDIR*CDTNY GOTO 120 1B0 RETURN END CCCCCC SIGNAL TI) OPERATOR FROM JOYSTICK SPEAKER• CCCCCC IEND DEFINES NUMBER OF BEEPS. SUBROUTINE BEEP{IEND) 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0085780/manifest

Comment

Related Items