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.

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., U n i v e r s i t y o f V i c t o r i a ,  A THESIS SUBMITTED  1977  IN PARTIAL FULFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in THE FACULTY OF GRADUATE STUDIES THE DEPARTMENT OF PHYSICS  We accept t h i s t h e s i s as conforming t o the r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA May 1983  (c) Bruce A l a n Faddegon, 1983  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the  requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e f o r reference  and study.  I further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s .  It is  understood t h a t copying o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain  s h a l l not be allowed without my  permission.  Department o f  PHYSICS  The  U n i v e r s i t y o f B r i t i s h Columbia  1956  Main Mall  Vancouver, Canada V6T 1Y3  Date  )E-6  (.3/81)  May 10, 1983.  written  ABSTRACT Since  Puck  and  Marcus  first  measured  the  effect  of  ionizing  radiation on mammalian c e l l s u r v i v a l i n 1956 the 'Puck p l a t i n g ' assay has been widely used as an endpoint f o r radiobiology experiments. has contributed greatly  to our knowledge of the processes involved when  radiation interacts with c e l l s , tissues, and whole animals. survival  experiments  (5-30 Gray)  where  un-irradiated Most  of  our  are carried  cell  cells.  survival  drops  primarily  accurate  due  below  50%  of  of  the  radiation  survival  of  Very l i t t l e data has been accumulated at lower doses.  knowledge  obtaining  Gener.ally c e l l  out at f a i r l y high doses  in  this  measurements made at higher doses. of  The assay  results  to the d i f f i c u l t y  c e l l s plated f o r the assay.  region  has  been  extrapolated  from  This i s i n part due to the d i f f i c u l t y  at  higher  survival  levels,  a  problem  i n accurately determining the number of  This s t a t i s t i c a l uncertainty becomes important  at s u r v i v a l l e v e l s greater than 50%.  The uncertainty can be s i g n i f i c a n t l y  reduced at these s u r v i v a l l e v e l s by accurately counting the number of c e l l s 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 i n t h i s t h e s i s .  With  the automated procedure the assay time i s reduced by a factor of three. In t h i s thesis the e f f e c t of the r a d i o s e n s i t i z e r misonidazole on c e l l s u r v i v a l was measured at radiation doses as low as one Gray. show  that  the  experimental  drug  is a  system used.  poor The  radiosensitizer  at  low  doses  c l i n i c a l and r a d i o b i o l o g i c a l  of these observations i s discussed.  The results f o r the  significance  This experiment exemplifies the value  of measuring c e l l s u r v i v a l at low doses. i i  ,  TABLE OF CONTENTS  PAGE Abstract  i i  Table o f contents  i i i  L i s t of tables  vi  L i s t of figures  v i i  L i s t of equations  viii  L i s t o f a b b r e v i a t i o n s and g l o s s a r y  o f terms  ix  Acknowledgements  . . . .x i  INTRODUCTION AND HISTORICAL REVIEW.  1  1.1  The s c i e n c e o f r a d i o b i o l o g y  1  1.2  The i n t e r a c t i o n o f i o n i z i n g r a d i a t i o n w i t h c e l l s  1  1.2.1  Q u a n t i f y i n g e f f e c t s w i t h the c o n v e n t i o n a l assay o f c e l l  survival 1.2.2  3  Measurements o f c e l l  s u r v i v a l a t low doses o f i o n i z i n g  radiation 1.2.3  Case i n p o i n t  7 f o r low dose s t u d i e s :  t h e oxygen e f f e c t  . . .  9  MATERIALS AND METHODS.  13  2.1  C e l l c u l t u r e 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 c o n v e n t i o n a l assay  16  2.3.2  The low dose assay  18  iii  TABLE OF CONTENTS (continued) PAGE 2.4  The automated  low dose assay system, ALDAS  2.4.1  ALDAS hardware  2.4.2  ALDAS s o f t w a r e  2.4.3  The c e l l  2.4.4  The c e l l c l a s s i f i c a t i o n program  20 22 •  l o c a t i n g program  27  LDALOC  31 LDACLASS  36  PURPOSE AND PROCEDURE.  41  3.1  Purpose o f the experiments  41  3.2  Experiment s e t A  45  3.2.1  Purpose and procedure  45  3.2.2  Choosing between t h e low dose and c o n v e n t i o n a l assays . . . 47  3.4.3  S u r v i v a l data  3.3  47  Experiment s e t B  48  3.3.1  M o t i v a t i o n f o r an improved  low dose assay procedure  . . . . 48  3.3.2  ALDAS and t h e low dose assay  50  3.3.3  Purpose and procedure  53  3.3.4  S u r v i v a l data  55  RESULTS AND DISCUSSION.  57  4.1  57  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 experimenter e r r o r  57  4.1.2  R a d i a t i o n dose r a t e and drug 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 .  4.1.3  S u r v i v a l assay u n c e r t a i n t i e s  59  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  63  iv  . 58  TABLE OF CONTENTS (continued) PAGE 4.2  4.3  Results  66  4.2.1  Method o f parameter e s t i m a t i o n  66  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  69  4.2.3  R e s u l t s o f parameter and c o n f i d e n c e e s t i m a t e s  72  Significance  o f the r e s u l t s  79  4.3.1  M i s o n i d a z o l e 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 o f the r e s u l t s  86  4.3.3  Radiobiological  4.3.4  s i g n i f i c a n c e o f 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  Suggested improvements t o the e x p e r i m e n t a l method  88  4.4  S u r v i v a l measurements a t low doses  90  4.5  Future research  92  4.6  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 between a s s a y s  4.5.2  Survival  4.5.3  O b s e r v a t i o n o f c e l l u l a r growth and d i v i s i o n  4.5.4  S u r v i v a l measurements a t low doses o f i o n i z i n g r a d i a t i o n .  dependence on oxygen t e n s i o n  Summary  93 93 . 93  94  BIBLIOGRAPHY.  APPENDIX.  . . . . 92  95  L i s t i n g s o f t h e ALDAS software r o u t i n e s  v  101  LIST OF TABLES  PAGE  I  L i s t o f t h e components o f ALDAS  28  II  N o r m a l i z e d , averaged,  III  Chi-squared f i t r e s u l t s  IV  Summary o f experiment  V  DMF a t s u r v i v a l l e v e l s o f 0.01, 0.1, 0.5, and 0.8, experiment  s u r v i v a l d a t a , experiment  Specifications  56  70  s e t s A and B r e s u l t s f o r a l p h a and b e t a . . . 82  set B  VI  set B  85  o f t h e automated low dose assay system, ALDAS . . . 91  vi  LIST OF FIGURES PAGE 1  Survival  curve showing t h e oxygen e f f e c t  5  2  Oxygen enhancement r a t i o a t low doses o f r a d i a t i o n  12  3  The f l a s k s used f o r growing  14  4  Diagram o f t h e i r r a d i a t i o n setup  15  5  Comparison o f t h e c e l l s u r v i v a l assay p r o c e d u r e s  17  6  LDA l o c a t i o n c h a r t  21  7  Photographs  23  8  Schematic  o f ALDAS  9  Schematic  o f t h e microscope  stage d r i v e r  26  10  I/O p o r t s on a n a l o g t o d i g i t a l c o n v e r t e r  29  11  LDALOC f l o w c h a r t  32  12  LDACLASS f l o w c h a r t  37  13  Time course o f experiments  14  Alpha and b e t a c o n f i d e n c e c o n t o u r s experiment  15  A l p h a and b e t a 70% c o n f i d e n c e c o n t o u r s experiment  set B  76  16  A l p h a and b e t a 95% c o n f i d e n c e c o n t o u r s experiment  set B  77  17  A l p h a and b e t a 99.9% c o n f i d e n c e c o n t o u r s , experiment  18  A l p h a and b e t a dependence on m i s o n i d a z o l e , experiment  s e t A. . . . 80  19  A l p h a and b e t a dependence on m i s o n i d a z o l e , experiment  s e t B. . . . 81  20  Survival  and i r r a d i a t i n g c e l l s  o f ALDAS  c u r v e s , experiment  24  set B  54  set B  vii  set A  75  s e t B . . . . 78  84  LIST OF EQUATIONS  PAGE  1  C o n v e n t i o n a l assay  survival calculation  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 d e v i a t i o n o f t h e b i n o m i a l d i s t r i b u t i o n  59  5  Low dose assay 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  60  6  Standard d e v i a t i o n o f t h e P o i s s o n d i s t r i b u t i o n  60  7  C o n v e n t i o n a l assay  61  8  C o n v e n t i o n a l assay 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  9  C o n v e n t i o n a l assay, s i m p l i f i c a t i o n o f t h e s t a n d a r d d e v i a t i o n  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 count  61  in survival 10  61  Low dose assay minimum 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 w i t h t h e u n c e r t a i n t y i n p l a t i n g e f f i c i e n c y t a k e n i n t o account  11  62  C o n v e n t i o n a l assay minimum 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 w i t h t h e u n c e r t a i n t y i n p l a t i n g e f f i c i e n c y t a k e n i n t o account  12  62  The r a t i o i n t h e number o f c e l l s t o be p l a t e d f o r the low dose and c o n v e n t i o n a l assays t o a c h i e v e the same a c c u r a c y i n s u r v i v a l measurement  13  63  E s t i m a t e i n t h e s t a n d a r d d e v i a t i o n o f s u r v i v a l from  survival  measurements 14  64  E s t i m a t e i n t h e s t a n d a r d d e v i a t i o n o f s u r v i v a l from u n c e r t a i n t y estimates  64  15  The c h i - s q u a r e d e q u a t i o n  65  16  The r e s i d u a l s  17  E s t i m a t e o f s t a n d a r d d e v i a t i o n o f s u r v i v a l from a s i n g l e measure-  18  (mathematical  definition)  65  ment o f s u r v i v a l  66  A s i m p l i f i e d chi-squared equation  68  vi i i  LIST OF ABBREVIATIONS AND GLOSSARY OF TERMS  A/D  Analog t o d i g i t a l c o n v e r s i o n .  ALPAS  The automated low dose assay system,  ASCAN  ALDAS s u b r o u t i n e used f o r moving t h e microscope 2.4.2.  CHO c e l l s Chinese hamster ovary  s e c t i o n 2.4. stage, s e c t i o n  cells.  c l a s s i f i c a t i o n day The day on which c e l l s a r e c l a s s i f i e d when u s i n g the low dose a s s a y . T h i s i s g e n e r a l l y 6-8 days f o l l o w i n g irradiation. coincidence The c l a s s i f i c a t i o n used f o r t h e low dose assay when two c o l o n i e s o v e r l a p on c l a s s i f i c a t i o n day. c o n v e n t i o n a l 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 e s t i m a t e d t h a t l e s s than 50% o f t h e c e l l s w i l l s u r v i v e . S e c t i o n 2.3.1. CVL  The c o n v e n t i o n a l assay.  D/A  D i g i t a l to analog conversion.  DMF  Dose m o d i f y i n g f a c t o r , d e f i n e d page 10.  FCS  Fetal calf  I/O  Input and o u t p u t .  killed  The c l a s s i f i c a t i o n used f o r t h e low dose assay when t h e c o l o n y formed by an i r r a d i a t e d c e l l a f t e r seven days o f i n c u b a t i o n has fewer than f i f t y c e l l s .  LDA  The low dose assay o f c e l l  LDACLASS  ALDAS program used f o r c l a s s i f y i n g c e l l s ,  LDALOC  ALDAS program used f o r l o c a t i n g c e l l s ,  LET  L i n e a r energy  serum.  survival. s e c t i o n 2.4.4.  s e c t i o n 2.4.3.  transfer.  l o c a t i o n day The day on which c e l l s a r e l o c a t e d when u s i n g t h e low dose a s s a y . T h i s i s g e n e r a l l y 2-3 days f o l l o w i n g 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 e s t i m a t e d t h a t g r e a t e r than 50% o f t h e c e l l s w i l l s u r v i v e . S e c t i o n 2.3.2.  LIST OF ABBREVIATIONS AND  GLOSSARY OF TERMS  (continued) MSCAN  ALDAS s u b r o u t i n e used f o r moving the microscope s t a g e ,  PER  Oxygen enhancement  possible  2.4.2.  section  ratio.  error 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 l o c a t e d on l o c a t i o n day cannot be found (or may have been d e b r i s which was m i s t a k e n 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 o f an i r r a d i a t e d c e l l which can grow i n t o a c o l o n y o f 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 b o t h t h e c o n v e n t i o n a l and t h e low dose assays.  TTL  Transistor-transistor logic. A c o n s i s t s o f a change i n v o l t a g e to zero V o l t s . An i n v e r t e d TTL f i v e t o z e r o V o l t s then back t o  x  l o g i c a l ( e l e c t r o n i c ) s i g n a l which from z e r o t o f i v e V o l t s t h e n back p u l s e i s a change i n v o l t a g e from five Volts.  ACKNOWLEDGEMENTS  I  gratefully  form of and  a two y e a r  the  support  provided field  acknowledge  for  National  provided  laboratory  would  like  to  e n e r g y he s o f r e e l y goals,  support  S c i e n c e s and E n g i n e e r i n g  by  the  materials  B.C.  Cancer  and  the  I  received  Research  Research  very  in  the  scholarship  Foundation  worthwile  Dr.  e x p r e s s my a p p r e c i a t i o n t o D r .  spent i n h e l p i n g to formulate  which  conference  sitting  I  on  could  encouragements  my  thesis  never  committee  have  offered  by  make e a c h d a y m e m o r a b l e the  fresh  contamination  cell  medium,  jokes,  the  succeeded my  if  special  understanding, our married me t o  family,  not  and  the  thesis,  Hoffmann and  the  friends,  and  warmth  and  humorous  a s s o c i a t e s who those  sterilized pipettes  and  f l a s k s , the  spilt  Dr.  cultures,  and  helped  responsible corny  especially  the  buns!  consideration  this beginning.  my  Geoff  all  over  the  insight.  without  I  Dr.  thank  laughter  a n d my m o t h e r  and  for  a n d c a r r y o u t my r e s e a r c h  reviewing  enjoyable.  I  thank  and p e r s e v e r a n c e d u r i n g  life  and  encouragement  d e p r e s s i o n - r e l i e v i n g cinnamon With  Branko P a l c i c  Lloyd Skarsgard for h i s scholarly c r i t i c i s m s ,  Cornelius Tobias f o r o f f e r i n g  for  fininancial  trips. I  for  the  my  very  who h a v e  her  so  love,  Cynthia Gayle W i t w i c k i John P e t e r  Faddegon  A u d r e y May  Faddegon  devotion,  n i n e months  lovingly  s p e c i a l people  thesis:  xi  for  t h i s f i r s t , wonderful  and f a t h e r  To t h e s e t h r e e  wife  I  of  encouraged  dedicate  this  1  INTRODUCTION AND HISTORICAL  THE SCIENCE OF RADIOBIQLQGY.  1.1  During science',  the  last  people  century,  have  radiations  which  are  subsequent  study  of  significant scientists  a  part  in  the  view the universe I  radiation  with  the  the  study  of  the  response  for  research  motivation constitutes important Second,  a health to  diagnosis.  This  instinct expand  our  can  is  Thus  class  of  and  treatment  of  everyday  living. of  lifestyles  most  It  is  of  detrimental  biological  and  in  this  effects used  of  as  most  effects.  of  tool  radiobiology  be has  the  First,  all  the  there  the  fulfilled become  an  of  -  radiation it  of  life. and  human  continue  important  is  radiation  natural  to  is The  treatment  effect is  benefit  exposure forms  in way  radiation.  medical  when  Third, to  on  for  effective  discovery  to  increased  radiation  a  in  Radiobiology  threefold.  age  and  resulted  and  of  particulate  l e a r n how we c a n  systems  is  'age  discovery  have  people  to  the  and  The  energy  important  radiobiology  of  electromagnetic  manifestations  radiations  sufficient  c a u s e i r r e v e r s i b l e damage However,  advancements  field  to of  OF I O N I Z I N G RADIATION WITH C E L L S .  that which c a r r i e s  death.  the  understood.  and  the  right.  THE INTERACTION the  of  of  be  well  horizons.  Perhaps is  be  pioneering  s c i e n c e i n i t s own 1.2  the  tool will  systems of  in  to  of  minimum o f  hazard,  evaluate  radiation  living  aware  these  changes  thanks  become  from  on  REVIEW  these human  energy  to c e l l s ,  radiations disease.  which pose to  the  ionize  greatest  molecules,  t i s s u e s , and a n i m a l s , are  also  Radiation  of  great  which  health  is  use  hazard  and w h i c h can  even r e s u l t i n g in  the  capable  of  in  diagnosis ionizing  2  molecules  is  appropriately  includes  X-rays  electromagnetic high  energy  most  dramatic  mutation  and  electrons, effects  t h e DNA o f  the  aberrations unable which and  to  cell  the  as  high  particles,  formation  which  known,  (Bacq and A l e x a n d e r ,  of  the  follow  the  1961;  cell  and  a great deal of  inflicted  and  treatment In  subsequently  by r a d i a t i o n order  scientists  to  have  strand breaks, organ  quantified to  in  cell  of  to  drugs,  effect.  the  the to  of  of  events  molecular  and  ions  of  the  of of  Whereas  1967;  cell  the  cells  that  organ  the  a biological  and W h i t m o r e ,  If  chromosomal  render  malfunction  as  general  system  Okada,  by  of  various  cellular  1970;  effects  on  biological  animals,  etc.  these  following  biological  endpoints  proliferative The  radiation, on  processes  1981).  radiation  cell  For example,  is  is  l o s s of  that  such  i n t h e m e c h a n i s m s b y w h i c h DNA damage  temperature, their  The  is  effects  whole  on.  as  there  modified  study  or  animal.  (Chapman a n d G i l l e s p i e ,  mutation,  death  terms  elucidate  observed  study  chosen  function,  combinations  interest  of  integrity  1 9 7 3 ) , many d e t a i l s h a v e y e t t o b e w o r k e d o u t .  currently  so  such  systems,  i n mutation  irradiation Elkind  and  of  c e l l u l a r biomolecules.  the  lead  death  pions,  the  can  forms  radiation  biological  physiology  radiation  energy  on  transform  organ  Such  particle  t h i s can r e s u l t  the  sickness or  of  try  in or  scheme  Hall,  events  well  radiations  eventually  a tissue  to  are  consequence o f  Changes  constitute  radiation.  i n d i r e c t l y c a u s e damage t o  can  divide.  as  alpha  these  i s damaged,  which  ultimately  protons,  are  which  (light),  of  which can d i r e c t l y or  ionizing  gamma-rays  radiation  and d e a t h ,  called  and  other  endpoints.  t a k e p l a c e between  the  such  capacity,  effects  The  systems as loss  of  of  various  agents goal  treatment  DNA  are is  and  to the  3  1.2.1 survival. a  Quantifying  effects  with  the  conventional  assay  used  endpoint  i n radiobiology  experiments  since  i t was  demonstrated i n mammalian c e l l s by Puck and Marcus i n 1956. determines  loss  of p r o l i f e r a t i v e  treatment such as i o n i z i n g  about one week under o p t i m a l plated  as a c o n t r o l .  Since  lose t h e i r p r o l i f e r a t i v e c e l l s which a r e a b l e the  one  week  considered lose  cell  The 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 , o r c e l l d e a t h , has been  widely  given  of  marginally  growth c o n d i t i o n s .  are c a l l e d  to divide  'killed'  colonies  results.  The l e v e l  them t o d i v i d e f o r  Untreated  of f i f t y  'survivors'.  to a  cells  are also eventually Single  o r more c e l l s A l l other  during  cells  are  Some m a r g i n a l s u r v i v i n g c o l o n i e s may e v e n t u a l l y  capacity  Generally  exposed  which d i v i d e i n i t i a l l y ,  t o grow i n t o c o l o n i e s  incubation  survive.  by p l a t i n g c e l l s  r a d i a t i o n and a l l o w i n g  some c e l l s  G e n e r a l l y , one  c a p a c i t y , an a r b i t r a r y c u t o f f i s chosen.  t o be ' k i l l e d ' .  their  capacity  first  f u r t h e r and w i l l may  continue  therefore  to slowly  d i e , while  divide  some  and t h e r f o r e  these m a r g i n a l c o l o n i e s do n o t s i g n i f i c a n t l y a f f e c t the of c e l l  s u r v i v a l f o r a given  treatment  i s d e f i n e d as  the p r o p o r t i o n o f t r e a t e d c e l l s which s u r v i v e r e l a t i v e t o t h e p r o p o r t i o n o f untreated be  c e l l s which s u r v i v e .  quantified  i n terms  In t h i s way t h e e f f e c t o f t h e treatment c a n  of c e l l  survival.  comparison o f t h e e f f e c t i v e n e s s o f d i f f e r e n t There  a r e many  radiobiologists: studies  related  radiation.  reasons  1. The c e l l  radiation  thereby  curing  this  provides  a method f o r  treatments.  endpoint  has become p o p u l a r  s u r v i v a l endpoint i s p a r t i c u l a r i l y  to radiotherapy,  Ionizing  proliferating,  why  This  the treatment i s used  to  the cancer.  o f cancer  prevent 2.  with  tumor  Experiments  among  useful i n ionizing  cells  from  performed i n  v i t r o a r e g e n e r a l l y e a s i e r and l e s s expensive than experiments performed i n vivo.  3.  Observations  from  cell  survival  experiments  can  often  be  4  extrapolated direction  to  for  in vivo research  understanding  the If  one  able  of  the  be  cell  Several the  cell  (Elkind  are  Survival  curves  of  cell  exponential  radiation follows:  in  can  energy  plotted  in  cell  The more damage the  reduced  transfer  deposited  per  In  to  paper  radiation  a  much  doses.  Because  1973). with  as  in  dose,  figure  generally  by  using  completely  exponentially)  followed  of  the of  track  s p a t i a l d i s t r i b u t i o n of  of  ionizing  a  die,  so t h a t  the  is  A few  1.  exhibit  sublethal  steeper,  damage  virtually  important  shoulder  of  length  of  the  radiation the  events  generally  This  survival is  primary  is  cell  defined ionizing  incident  explained of  the  as  cell,  radiations often  curve.  from a given energy  the r a d i a t i o n  the  site  sparsely ionizing  radiations.  given  caused by  sensitive  ionizing  ionizing  c h a r a c t e r i z e d b y t h e LET o f  events  This  accumulated near  cell will  (LET)  unit  Hall,  1970;  semi-log  with higher  observed  d i s c u s s e d more  accumulation of  survival.  as d e n s e l y  width  modification  endpoints) .  been  represent  survival  after  relevance to t h i s t h e s i s are discussed here:  affect  as e f f e c t i v e a  generally  exposed  in  understood,  a  other  have  are  (almost on  of  aid  death  sufficiently effect  effects  rapidly  repair,  cell  to  also  and  assay.  very  can  lead  (and  Okada,  cells  can  survival  effects  motivation  endpoint  the  1976;  spatial distribution  t h e more l i k e l y not  These  provide  are  predict  cell  thought to  decrease  the events  biological  mammalian  s u r v i v a l e f f e c t s of 1. The  eventually  and Whitmore,  shoulder,  This  i s r e l a t i v e l y easy to  decreases  curves  the  on  endpoint.  survival  which  cell  interesting  survival  an i n i t i a l  a  survival  survival  which  quantitatively  of  very  elsewhere. cell  to  thereby  4.  t h e mechanisms o f  treatment  addition,  and  in vivo.  events  irradiation. should  studies  as  are  reflected The  linear  the  energy  particle. of  The  radiation  as w e l l as by t h e d i m e n s i o n s  of  is  5  T  1  I  1 CHO  CELLS  SUMMATION  2  o  (1979-1981)  1.0  r-  o < cc  0.1  C5  > > cc 3  0.01  0.001  \  CO  0  5  10 D O S E  Figure 1:  o  2  15  20  25  30  ( G r a y )  SURVIVAL CURVE SHOWING THE OXYGEN EFFECT.  Exponentially growing CHO c e l l s were i r r a d i a t e d i n dilute suspensions at 0°C under aerobic (O?)' hypoxic {H^) conditions (x-ray source: Picker, 280 Kvpf. C e l l s were assayed for s u r v i v a l . The c e l l s i r r a d i a t e d under aerobic conditions are much more sensitive than those irradiated under hypoxic conditions, with an OER of about 2.8 at a survival l e v e l of 0.01. (Reprinted with permission from P a l c i c , Brosing, and Skarsgard, 1982). o  r  6  the  radiation  track,  the  ionizing  radiation  will  significantly  not  the  so c a l l e d  of  after  the  cell  which  an  region  1  cycle.  This  cells  c a n be  replication  has l i t t l e  3. grow  In  These  optimal  experiments  radiation between period  is  (Elkind  show  than  in  called  long  ratio  containing  1  of  the  survival  l e v e l of  For  cells  This  is  with  LET's  of  function  1970).  and  on t h e i r the  during  the  location  importance  mitosis.  are  in  of  the  radiation  If  irradiated,  a while, more  then  the  DNA  in  the  likely that  sensitive repair  to  to  survive  damage  cells)  (hypoxic  are  supported  enough t h a t  enhancement  ratio  generally  cells).  is  to  This  OER i s  after  dose dose  more  (Figure 1,  this  ratio  from P a l c i c  is  rate  rates.  sensitive  effect,  defined  approximately  et a l ,  1982.)  a  place.  the  as  3.0  Thus,  so  defined  t o the dose w i t h oxygen t o y i e l d the  cells  the  repaired  a s p e c i a l t e r m was  (OER).  again.  take  by  to  if  irradiation  processes  is  allowed  irradiated  a r e more s e n s i t i v e t o h i g h e r (oxic  oxygen  cells  evidence  oxygen  mammalian  0.01.  for  i s important  dose w i t h o u t  level.  dying.  LET i s a c o m p l e x  indicating  less  This  oxygen  the oxygen  survival  are  become  enough  little  'oxygen e f f e c t , it:  cells  1959).  containing  to quantify  ionizations  r e p l i c a t i n g then information  for  fractions,  Cells  Sutton,  Cells  cells  stage  conditions  e x p e r i m e n t s w h i c h show t h a t 4.  cell  radiations  e x p l a i n e d by  experiments  that  delivered  incubation and  dose  growth  irradiations. of  of  densely  replication.  fractionated  under  very  d i s p l a y maximum s e n s i t i v i t y t o  t i m e t o r e p a i r damage b e f o r e  DNA may b e l o s t d u r i n g  the  for  for  density  r a d i a t i o n depends  generally  immediately  the  In g e n e r a l ,  to  Most c e l l s  the  in  a point  l i k e l i h o o d of  on L E T ,  p r o c e s s o f DNA r e p a i r . before  is  which occurs  (ICRU r e p o r t  s e n s i t i v i t y of  There  increase  200 k e V / u m o r g r e a t e r .  r a d i a t i o n energy. 2 . The  core.  increase the  'overkill  approximately  track  the same at  in  a  the  7  presence of the  oxygen,  absence  of  a cell  oxygen  needs o n l y  to  result  b e i n g k i l l e d as a consequence interact  with  of  radiation  one t h i r d  in  of  ninety-nine  irradiation!  to  decrease  the  out  of  Agents cell  dose  it  one  requires  in  hundred  cells  such as oxygen  which  survival  are  called  radiosensitizers. 1.2.2  Measurements  radiation. levels  of  Most of about  survivals  at  the  0.5  vitro  studies  these  results  on  hand,  s i x weeks. normally  enough  to  radiotherapy those  doses  used  a t low doses of the  effects  survival nature repair  of in  doses  for  2.  at  is  the  now a m p l e 1.  For  radiotherapy. daily  low  doses  literature  to  cells  Generally  doses of  studies Gy  of  1.0  cell  in  Thus,  to  to  measures for  the  vitro  studies  doses  (high c e l l low  lesion are  survival)  survival basic in  predictions  Low d o s e m e a s u r e m e n t s  cell  concepts the  in  vivo.  the  the  of  cell  of Some  (for  treatment  radiation  of  also  large  used  degree  survival  of  than  Measurements  at  that high  such  as  the  importance  of  DNA  Models of  in  lower  observed  radiobiology  of  other  effects  survival.  of of  for periods  of magnitude  are  in  treatment  3 . 0 Gy  of  controversial. of  measuring  m u s t b e made t o c o n f i r m  levels  DNA a n d  still  of  survival  results  produce  a r e a s much a s a n o r d e r  in  ionizing  i n a c t i v i a t i o n , on t h e  order  the  of  motivation  decades  extrapolated  5-30  Certain  death  c o n c e p t s make v a r i o u s radiation.  the  critical  cell  of  fraction)  observed  the  to  accurately.  radiation  levels.  in  at  p a l l i a t i v e doses a r e sometimes h i g h e r )  use  (per  been  Laboratory  measure  data  levels:  involves  with curative intent,  survival  There  have  applied  cancer w i t h r a d i a t i o n  two t o  less.  survival  cells  are  cell  survival  or  higher  of  cells  based at  s u r v i v a l may h e l p t o  on  such  low doses  of  distinguish  8  between  these  underlying few g r y s  models  and  assumptions.  to  determine  Many  feel that  constitute  t h e most  sixth L.H.  Gray  important  the  relative  the  response  aspect of  importance of  cell  cells  of  their  to the  killing  (eg.,  first Alper,  1979). The survival at  low d o s e s .  low doses i s  the If  at  major  conference (Alper,  important,  reason  is  the  a c c u r a t e measurements  order  to  collect  w o u l d be v e r y experiments, Even  if  the  are  errors  sources of  the  from  error  the  volume  sample  a  large  the  eliminated  by  estimating  this  the p l a t i n g  the  sample  of  accurately number.  e f f i c i e n c y of  Perhaps  cells  cells an  variation are  the  counting  The the  first number  In p r a c t i c e t h i s w i l l the  experiment,  the  there  are  two These  as  estimated  accurately  measured  in  the  proportion  of  survive.  In  to  surviving  plated  cells  c a n o n l y be r e d u c e d  cells  also  jeopardy.  significant  plated  uncertainty of  in  survival  survival.  'destined'  and  in  no  uncertainties  c a n be  plated,  number o f  by  virtually instead  significantly  relative  in  experiments  factor  cell  data.  procedures, the  with  The s e c o n d u n c e r t a i n t y cells.  survival  w o u l d be  assay of  that  cell  a crucial  perfectly  statistical  plated  data available?  statistics  from  cell  survival  experiments  plated  to  cell  in  of  conventional  number o f  sample volume the  is  measurement  in  measurement  reasonable  conventional  and t h e  both  number  for  1974 was d e v o t e d  inherent  Since time  minimum  in  If  using  performed  have a P o i s s o n d i s t r i b u t i o n . plating  be made  data  in  so l i t t l e  are  the  cells  there  such lengthy  within this plated  a w e l l mixed  to  variations of  1974)  of  and  statistical  c o n c e n t r a t i o n ,of cells  results  London  uncertainty  consuming.  experiments  experimenter major  are  sufficient  time  ed,  why i s  large  in  of  increase untreated  9  cells  that  survive,  along with c e l l s in  a  sample.  uncertainty The a  for  to  could  be  by  be  the  conventional  is  possible  to  do  most  the  of  uncertainty surviving  in  the  cells plated  of  this  assay.  Case i n  oxygen  helped to death.  in this  to  be  debris cells  decrease  the  lost  Thus  measuring  the  This  at  least  cells  plated.  a binomial  uncertainties  In  fact  the  total  This  increases  the  discussed  more  the  number  since the  it  could  reduces  distribution  are  of  alternative  a computer  addition,  further  cells  degree  levels.  that  cell  accuracy few  some  significantly  In  in  a viable  survival  assay  gain  of  survival.  comparitively  s u c h an e x t e n t  quickly. of  high  for  t i r i n g were t h e  and  since  time.  t h e s i s , makes u s e  assayed  t o make t h i s p r o c e d u r e  point  effect  has  for  low dose  added  to  The o x y g e n e f f e c t may h a v e  regions is  (cannot  further  and e x t r e m e l y  studies:  our  of  complication in  hypoxic  concern  cells. that  proliferate),  the  of  number  accuracy  completely  although the  of  oxygen  effect.  in  radiobiology  Study and  i n the events which lead to  important treatment  When a t u m o r  the  knowledge  s o r t o u t t h e mechanisms i n v o l v e d  One p o s s i b l e  there  be  a c c u r a t e l y known. These  will  manually,  assay to  number has  count  4.1.  1.2.3 the  is  for  the  very  plated  cells  completely would  to  cells  consuming  in order  assay  work  is referred  reasonable  automate  of  section  a  i s required  to  time  cells  in  generally  the concentration of  efficiency  plated  performed  located  automation  of  locating  locating  counters  survival.  as i t  a s s a y w o u l d be v e r y  obtained  plating  i n the measured assay,  cell  i n an o v e r e s t i m a t e o f  increased  low dose  locations  automatic  resulting  An  microscope  This  since  is  consequences f o r of  tumors  is  that  well  oxygenated  cells  are  radiation  will  have  effect  on  less  cell  radiotherapy. they  treated with ionizing  the  has  contain radiation  inactivated the  hypoxic  10  cells,  and  resulting  the  survivors  i n regrowth  Since valuable  hypoxic  to  study  sensitizing  believed  to  electron  of  the  drug  to  et  al,  al,  1976; a  has  be  cells. also  the  OER.  a  The  same  Denekamp  et  al,  Misonidazole  of  survival  in  1973). They  It  their  about  1974  2.3  at  1976).  (Denekamp  This one  has  been  in  1973;  as  a and  et  al,  institutions,  cells  is  of  oxic  et  1975).  be  about  is  1974).  in  vivo.  Currently,  more  at  lower  (Moore  h a s a l s o b e e n shown t o 1975;  are already  including  a 2.0  and  al,  radiosensiter  and  the  first  to  radiosensitizer  1974  by  (DMF).  the  cells,  Asguith  to  analagous  shown  15 mM c o n c e n t r a t i o n . It  The  factor  w i t h a maximum DMF o f  act  high  affinity.)  was  effect  are  t o t h e dose i n  level.  a n d a f e w r e l a t e d compounds of  oxygen  radiosensitization  the drug  which i s a b e t t e r  Adams e t a l ,  t r i a l s a t a number  of  of  dose m o d i f y i n g  and W i l l s o n , to  vivo.  their  cells  shown  in  be  1977).  electron  degree  detail.  been  1976;  would  (Adams,  as a r e s u l t  high  measurable  (Foster  effect  it  s e n s i t i v i t y of w e l l oxygenated  in  no  treatment  (Wardman,  radiation  eventually  1955).  radiation.  Metronidazole  studied  a DMF o f  tumor  the dose w i t h o u t  to misonidazole  radiosensitizer  clinical  has  h y p o x i c mammalian  having  McNally,  1980).  also  1977.)  has  and G r a y ,  drugs.  to  yield  of  1974;  i s given  concentrations  as  of  hypox i c  attention  al,  to  mM c o n c e n t r a t i o n ,  Metronidazole (Begg  of  cells  r a t i o of  in  ionizing  class  Wardman,  definition  to  to  a  reoxygenation,  which can mimic the  increase the  as the  radiosensitizer  toxic  problem  c a n be e x p r e s s e d i n t e r m s o f  nitroimidazoles  10  such  do n o t  DMF i s d e f i n e d  at  cells  (Oxygen  (e.g.  these drugs  the  a  upon  (Thomlinson  radiosensitizers  affinity.  presence  are  s e n s i t i z e hypoxic  radiation.  to  cells  are  nitroimidazoles  proliferate  of the tumor.  hypoxic  Nitroimidazoles  will  the A.M.  Turner  et act et  undergoing Evans  11  Clinic  i n Vancouver,  B r i t i s h Colunubia.  Many s t u d i e s  appears t o r a d i o s e n s i t i z e hypoxic tumors  i n humans.  et  A good  al,  1977;  given  in  Thomlinson  the  Sensitizers", There regions findings ability  (L.W.  Brady,  some  tumors  at  the  useful  of  of  Biscayne  whether  with  (Palcic  oxygen on  of  this  Urtasun  subject  Conference  the  i s measured t o be a b o u t corresponding  itself  et  has  al,  (of  of  hypoxic  Indeed,  limited  recent  radiosensitizing  the order  1982.)  hamster  of  two  survival  2  measure  shows  the by  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  of  survival of  about  l e s s t h a n one  Gray.  poor  indicated  that  misonidazole  doses.  (Durand  in vitro  at  and  misonidazole  on  radiation  low  the as  produced  by  If  observation  this  explain (e.g.  the  Brown,  low doses?  as  doses?  Dische,  1980).  is  be  a poor What  of  Gray. like  Chinese It  was  oxygen,  representative  success  of  dose  doses,  studies  radiosensitizer is  study measures  survival  misonidazole,  minimal  may  1980).  This  one  Fractionated  low  or  n i t r o i m i d a z o l e s which mimic the r a d i o s e n s i t i z i n g e f f e c t of oxygen a l s o low  at  0.8  the  at  radiosensitizer  l e v e l s of  are  radiosensitizers  poor  Figure  Gray)  as measured  1.5±0.7 f o r  cell  t o doses t o o x i c c e l l s a  is  "Radiation  treatment  radiosensitizers.  radiation  Chinese  oxygen  is  to  oxygen  For the experimental  oxygen  review  1978;  s e n s i t i z a t i o n at these doses i s d i f f i c u l t t o  et a l .  If  as  doses of  Palcic  greater,  Key  (Dische,  misonidazole  1980).  improved  techniques.  effect  of  indicate that  the degree  measured  1976).  controversy  clinically  current  al,  ed.,  c a n be  in vitro  although with  proceedings  is  in  et  show t h a t  its the  that  misonidazole  the in  vivo  at  low  ability  radiosensitizing effect  diminishes of  already  radiosensitizing  hamster  found  in  have  ovary the at  clinical patient  cells  at  doses  of of  radio-sensitization low  radiation  situation, treatments  dose. it  to  may date  12  0.4  0.6  0.8  1  D O S E (Gy) IN 0  Figure 2:  6  8  10  2  OXYGEN ENHANCEMENT RATIO AT LOW DOSES OF RADIATION.  Exponentially growing CHO c e l l s were i r r a d i a t e d i n dilute suspensions a t 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 s u r v i v a l data obtained a t 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 c l e a r l y dose dependent f o r t h i s experimental system. (Reprinted from P a l c i c , Brosing, and Skarsgard, 1982 with permission.)  13  MATERIALS AND METHODS  2.1  C E L L CULTURE The  Chinese ovary  in  vitro  hamster  (CHO)  high  calf  lines  line  (FCS),  to  and  in  can  culture  in  tissue it  be  here  culture.  grows  grown  alpha  antibiotics,  grown i n a s p i n n e r regulated  grown  reported  were  The  rapidly,  in  performed  Chinese  monolayer,  flask,  7.4  by  medium,  shown i n f i g u r e  continuous  3,  gassing  buffer.  with  The  5%  CO^  in  and  We g r e w CHO 10%  fetal  cultures  i n an i n c u b a t o r a t  with  with  suspension,  supplemented  and b i c a r b o n a t e  on  hamster  can be c l o n e d  (Thompson, i n J a k o b y and P a s t r i n a , e d . , 1 9 7 9 ) .  suspension  serum  experiments  i s widely used:  efficiency,  s o f t agar t i s s u e in  biological  cell  cell  plating  cells  PROCEDURES.  were  37°C; pH  air.  The  is  cell  4 culture  is  asynchronous 2.2  diluted  about  7X10  e x p o n e n t i a l growth w i t h a doubling  source  H V L = 1 . 7 mm C u ,  of at  irradiating  irradiation  radiation  a dose  following approach,  the  1.2x10^ one  of  directly  on  the  head  the  X-ray  dose  figure  4.  delivered  of  1969;  in  cells/ml,  time of  the  head the  maintaining  about  12  hours.  The m e a s u r e m e n t  of  2.0  machine,  Gray  per  Thomson a n d R a u t h ,  from  culture  vessel  above  the  were time  X-ray  to  The  the  cells.  0.3  3,  cells  and  Picker  figure  distances  and  a  between  cells/ml.  two  to  was  Cells  shown  at  above  rate  cells:  platform  vessel  used  (Parker et a l ,  vessel,  approximately  the  to  I R R A D I A T I O N PROCEDURES. The  for  daily  in  were  head  irradiated. taken  The  the dose r a t e  for  minute.  The  removed  on  a  the  X-ray  The  height  irradiation  irradiation  to  suspension  placed of  KVP,  1 9 7 4 ) , was u s e d  dilute  was  270  set-up  is  the of  wooden tube of  or the  determined shown  is discussed in section  in  4.1.2.  STIR  BAR  I  Figure 3:  THE FLASKS USED FOR GROWING AND  Gas  F Io w  IRRADIATING CELLS.  Cells are grown i n suspension culture i n alpha medium supplemented with 10% FCS, a n t i b i o t i c s , and bicarbonate b u f f e r . Cultures are incubated at 37°C i n spinner f l a s k s (a); pH i s regulated to 7.4 by continuous gassing with 5% CO^ i n a i r . C e l l s are i r r a d i a t e d i n i r r a d i a t i o n vessels (b) with continuous^ nitrogen flow over the s t i r r e d 15-20 ml suspension of 1.2 x 10 c e l l s / m l . Samples were obtained by removing the o u t l e t stopper b r i e f l y and lowering a pipet down the neck of the v e s s e l into the suspension. A l l experiments were performed at O'C.  15  G  plexiglass  A  S  O  U  T  container  C  E  L  L  S  U  S  P  E  N  S  I  O  N  ice water  X  when x = 0.0 cm dose rate =1.6 when x dose when x dose  Gy/min  = 10.0 cm rate =0.8 Gy/min = 49.0 cm rate = 0.3 Gy/min  20 X 20 era colimator  X-ray head  Figure 4:  DIAGRAM OF THE IRRADIATION SET-UP.  The i r r a d i a t i o n vessel (figure 3) was placed i n a p l e x i g l a s s i c e water bath. A s t i r motor was suspended above the v e s s e l . To obtain a dose -rate of 1.6 Gy/min the p l e x i g l a s s container was placed d i r e c t l y on top of the 20 x 20 cm colimator. To achieve a lower dose rate of 0.8 and 0.3 Gy/min the p l e x i g l a s s container was supported around the edges by a wooden platform 10 cm and 49 cm above the colimator r e s p e c t i v e l y i  16  2.3  C E L L SURVIVAL A S S A Y S . The  irradiated  methods:  the  samples  conventional  were  assay or  diagramatically represented 2.3.1 than  were  plating' the  assayed  assay.  sample  of  cells  was  Marcus,  the  Adams, of  damage  1977;  cells  in  c a u s e d by  Palcic  et  each sample  Coulter  Electronics  contain  enough  plated  in  cells  remove  (Moore e t  for  A volume  dishes  of  colonies in  5ml o f  and grown a t  5%  for  time  cells  flow  are  7  to  at  which  s t a i n e d w i t h methylene  colony are d e f i n e d judged  days,  have  'non-survivors'  as  lost or  their  'killed'.  Survival,  blue.  'survivors',  S = C_ NP  These  of  two  assays  are  survival  after  to  the  is  The  'Puck  This  grown  under  interact  Stratford  and  concentration  estimated using sample  50  cells  or  more  to are  10% F C S ,  37°C i n a n i n c u b a t o r is  the  estimated  a l p h a medium w i t h  medium  is  discarded  with  and  the  50 c e l l s  per  l e s s t h a n 50 c e l l s p e r c o l o n y  are  proliferative Survival  1976;  each  Colonies of  and o f  less  irradiation  cells  1981).  of  of  conventional  al,  survival  and b i c a r b o n a t e b u f f e r ,  2  one  misonidazole.  toxic  antibiotics, C0  the  the  Korbelik et a l ,  100-200  petris  by  t h i s t o x i c i t y h a s b e e n shown t o  assayed  give  assay.  Immediately  shown t o b e  counter.  to  5 cm d i a m e t e r  1978;  t o be  cell  to  irradiation.  al,  survival  using  1956).  has been  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 , with  low dose  survival  centrifuged  done b e c a u s e m i s o n i d a z o l e  cell  C e l l s with estimated  cell  and  for  5.  assay.  for  (Puck  the  in figure  The c o n v e n t i o n a l  0.5  assayed  more t h a n  capacity  i s e s t i m a t e d as  and  are  defined  follows:  Equation  C=number o f s u r v i v o r s (colonies) 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 dose ( p l a t i n g  1  efficiency)  as  17  TH£  CONVENTIONAL ASSAY  TH£  irra J-io.'teJ..  are.  Ceils are ,ce.n. iri fu< rt.stispe.nJ.eJL.  LOU POS£ f\SSt\Y  comptftcr scan, reference . point.  0000006% OOOOOOO OOOOOOO OOOOOOO OOOOOOO  tricretcst pCo."te. uoih. 1G> O-'rmttJcU.s  boliont vietJ, 2.Sc*J-fUsK.  /Ipproximateiu 20o/S ceU,s 300-600 cells are plated, per vessel. are eC-a.te.cL 2V ecich or Centri.Tu.qpjcIon. is not required, since. thrte pevris dUsheSjuhe, ceCLs are HiLated. 2ooo~£x>oo times. 'survival*. SiS the. estimated. Sarvl araJLaa&e.  h  '£ud.e«t  mero-hle.  Cells a-re. Cocojt&cL tolih a. Microscope. #u.tot«a£io/l uitLL eventILO-LLLI replace. tne.jra.da.ate. stu-dentr.  cms Joystick -to t*K.troL s ia*e motion. Ce,U S are  tncubaieoL for six to ci^ki  dUas after  pLo-tina^. Cetls cakt'ek.  oUvide  After medium weerh  oiipco.rdedjee.tts  it trie re.  £Man  are couttieeL.  Figure 5:  fO  ct-CCs.  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. '  COMPARISON OF THE CELL SURVIVAL ASSAYS.  Two d i f f e r e n t procedures were used f o r assaying c e l l s f o r loss of proliferative ability, the cell survival assays. The conventional assay i s used f o r c e l l s estimated to have a s u r v i v a l of less than 50% . The low dose assay i s used f o r c e l l s with s u r v i v a l of greater than 50%.  18  2.3.2 than  The  0.5  low dose  were  Irradiated  assayed  samples  a  level  100-400  which  cells  microscope. of  the  If  by  entire  wanderers.  in  prevent  well  the  since  'low  samples  (Korbelik suitable  et  al,  vessel  1981) . for  greater  dose  assay'.  were  diluted  reducing the concentration of  a  must  be  wandering  misonidazole  An  aliquot  viewing  under  from  per  25 c m filled  microtest  culture  with  medium  their  locations  radiation.  Six  is  to  'survivors'  only  motion  respective  days  Colonies  50 m l )  plated two  in  or  following with  prevent  used  (about  cells  200 u l o f  to  are  recorded  eight  re-examined.  5)  plate)  flasks  and  as  aid  2  their  to  three  medium  5)  vessels days  than  a n d 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  the  eliminate  each  50  are  are  after  irradiation  greater  of  problems  (figure  the  of the  ( v e s s e l c o n t a i n i n g an 8x12 a r r a y  a microscope,  location  are defined  from  of  located to  cells  (20 m l If  With the  coordinate  as  to  wanderers.  coordinate  centrifuged  toxic.  plated  per  flask  exposure  not  using  10 mm d e e p w e l l s a s shown i n f i g u r e  required  caused  not  survival  m i c r o t e s t p l a t e s are used  7 mm d i a m e t e r , are  cell  of  S u f f i c i e n t medium m u s t b e a d d e d t o t h e v e s s e l t o r e d u c e  medium  colonies.  C e l l s with estimated survival  to plating,  is  is  for  were  2000-5000 f o l d p r i o r to  assay.  cells defined  'killed'. On  day,  only  potentially viable  cells  are recorded;  are not recorded.  This  which  location  is  taken  into  those  locations is,  lysed cells  i s s u b j e c t to a c e r t a i n degree of account  l o c a t i o n s w h i c h h a v e no v i a b l e  as  follows:  cells  on  debris  experimenter  bias  a r e c l a s s i f i e d as  'possible error'  and  the  recorded  and c e l l  day  returns  locations  represent  classification  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  to  the  constitute to  that  considered  On c l a s s i f i c a t i o n d a y t h e the  location  day  and  observer  classifies  the  19  colonies  as  'possible  'survivors',  error'.  e i t h e r one o f the  two  of  the  were  nearby  its  should  not  be  if  cells  the  relative proportion  of  It  identify  difficult  to  about  100 um o f  another  employed.) plates cells  There  if  more t h a n  per  well) .  plated in If  25 c m a  day  is  colonies  are  location  errors.  colonies low  of  the  could or  counted  the  Thus  low  only  the  as  there  'possible dead  number o f  a few c e l l s  1  (less  than  no  error'.  0.5)  if  of  plate  recorded  cells of  in  the  within  microtest  cells  two were  the  other  are  (equation  number  10% o f  colony  'possible  half  2)  location  that  plated  when  is  in  and  classifications  least  it  (average of  error'  (at  than  of  avoided.  Half  The  grown  c o i n c i d e n c e was  automated  the  the  be  colonies  less  coincidences  was  viable  colonies.  significant  'radius  of  at  colonies,  cells  error  found  were  'possible  'possible  levels  be  to  (If  proportion  be  of  a r e r e c o r d e d as c o i n c i d e n t .  a 100 um  assay  the  of  cells.  generally  relative  were p l a t e d p e r  dose  few  survival.  can  generally  that  number  cells  not  if  The  was g e n e r a l l y  survival  200  c l a s s i f i e d as  in  of  significant  about  will  used  c o l o n y would have  the  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  2  location  uncertainty  a  either  centre  is  a  cell  or  diameters)  non-proliferating,  cells  a colony  the  of  colonies  Otherwise  coincident  colony.  After  colony  classification  as h a l f  was  was  non-surviving  is  If  colony,  definition  c a l c u l a t i o n of  overlap.  if  cell  colonies  separate  neighbour  as  This  over  the  coincident.  recorded  another  (about 5-10  colony.  the  even though they  considered  surviving  in  proliferate,  neighbour  100 um  colonies  included  with  considered coincident  neighbouring  surviving  cells  distinguished over  Two c o l o n i e s a r e  favouring  Coincidences  'coincident'  the colonies i s w i t h i n  perimeter  prevent  'killed',  the  result  a c c u r a t e measurement  error'  in  error'  located) of  an  estimated  'possible cells  the  considered  which i s of  on  at  survival  20  is  not as i m p o r t a n t .  Survival,  Survival  S =  i s estimated as f o l l o w s :  C NP  Equation 2  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  For experiment system  was  efficiency).  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 .  unfortunately  c l a s s i f i e d manually  not  available  and  cells  through the microscope using  6 i s a t y p i c a l l o c a t i o n c h a r t used before  The a u t o m a t e d  were  located  a location chart.  and  Figure  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  For  experiment  the  low dose  detail  in  control  set B cells assay  section  a motor  were p l a t e d  procedure 2.4.  driven  used  i n 25 cm  for  flasks.  experiment  F o r the improved  procedure  microscope  to  stage  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 .  Improvements  to  set B are described  in  a computer  a i d the  i s used  experimenter  The l o w dose  in  to the  assay i s discussed  i n c o n s i d e r a b l y more d e t a i l b y 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 This  at  section describes  the B r i t i s h  Inspiration Bruby  Columbia  for  for  Cancer  and s o f t w a r e  Research  Centre,  of  s y s t e m came  this  and D r . Tolmach o f Washington  Missouri,  time  School of Medicine,  lapse  (R.  Heye e t a l , 1 9 8 1 ) ,  photography.  Stanford,  in part Dr.  for  B.C.  ALDAS  (BCCRC).  from D r s .  L.  R. K a l l m a n o f t h e  California,  University who h a v e  developed  Vancouver,  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 , Washington,  1980),  Louis,  the hardware  the development  Stanford University al,  (ALDAS)  (H. Kemper e t  School of Medecine,  developed  similar  St.  systems  21  Figure 6:  LOW  DOSE ASSAY LOCATION CHART.  This a the chart used to record the locations and c l a s s i f i c a t i o n s of the c e l l s assayed f o r s u r v i v a l with the low dose assay. Irradiated c e l l s were placed i n microtest p l a t e s (figure 5) and located under the microscope 2-3 days following i r r a d i a t i o n . Each dot (") on the chart corresponds to a located c e l l . Cell locations were re-examined and c e l l c l a s s i f i c a t i o n s were recorded on the chart 6-8 days following i r r a d i a t i o n . The symbols on the chart have the following meanings: C-'survivor', a colony with more than 50 c e l l s ; D - ' k i l l e d ' , a colony with less than 50 c e l l s , • -'possible error', a c e l l cannot be found at the recorded l o c a t i o n , and /^-'coincidence', colonies have overlapped.  22  2.4.1 basic The a  ALDAS h a r d w a r e .  system hardware  core of  ALDAS  television  high one  recording  f r o m image  facilities  stage  motors which  must  will  attached  translates of  the  that the  the  stage.  there nut.  steps.  If  which  a l o s s of  and  built  by  Semprex  In  adapted  movement p e r  order  to  to  our  purposes  primarily  motion  the  100  of  camera.  10  for of  ALDAS t h e  The  figure  8.  mounting such  advantages  construction,  and  a  that  the  friction  case be  they  linked  image  developed  t o have  stage.  the  stage  um r e p e a t a b i l i t y  than it  the  lead  s i n c e any  Division  um o r  the  nut  motion  10 s t e p s been  The  and miss  Campbell,  Product  have  so  designed  stage  same d i r e c t i o n ;  satisfactory.  l i n k between the  This  'looseness'  Street,  Motion  would  from the  nut  screw  lead screws p r o v i d i n g 100  A  stage  s t a g e was  modify  American  stepping  f r i c t i o n they w i l l  stage  a machinist  less  was  the  587  microscope.  locations  between this  the  build.  translational  The m i c r o s c o p e  corporation,  i n the  the  to  steps.  mounted on  repeatability.  um r e p e a t a b i l i t y  to  miss  precision  attached to  step with  may  that  be  cannot e a s i l y overcome  our  so  the  friction  firmly  s y s t e m component  screws to  screws must  recorded  to a looseness  in  Although  the  a r e mounted on t h e  motor  achieve  always approach  screw must  was n e c e s s a r y  step stepping motors stage  lead  a minimum o f  in  be  sturdiness  in  result  could  a requirement  overloaded,  will  it  not  minimize  the motors  system.  schematically  and p o l a r o i d  to  lead  this  significant.  rotational  It  shown  smoothly  The n u t m u s t b e  California.  is  of  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  quality,  each  The  is  below  a photograph  s t a g e was t h e most d i f f i c u l t  be to  is  are  glide  not  7 is  35 mm c a m e r a ,  microscope  The m i c r o s c o p e The  described a ZEISS  camera,  quality obtains  is  Figure  so  that  1.8°  per  a 10 um  backlash.  necessary  to  however  for  b a c k l a s h was  due  l e a d screw nut and  the  23  .  Figure 7 : PHOTOGRAPHS OF ALDAS. These photographs show the hardware components o f the automated low dose assay system.  24  Com.pu.i&r  joystick.  spco-kcr  'iou$hcck a-neL buJrtons J°<j•stick  Q  Microscope.  s k o L j e ,  :ors  + Q s i f A <> e  F i g u r e 8:  micro  swi •ic-hc s  SCHEMATIC OF ALDAS.  The f o l l o w i n g i s a t y p i c a l sequence o f e v e n t s : The computer sends an a n a l o g s i g n a l t o t h e j o y s t i c k speaker through the D+7AI/0 board t o s i g n a l t h e experimenter w i t h an a u d i b l e 'beep' that t h e microscope stage i s i n his/her control. The experimenter, w i s h i n g t o move t h e s t a g e , moves t h e j o y s t i c k which sends a d i g i t a l s i g n a l t o t h e computer through t h e D+7AI/0 board which r e p r e s e n t s t h e magnitude o f t h e speed t o move the s t a g e . The computer moves t h e 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) p u l s e s a t t h e a p p r o p r i a t e f r e q u e n c y t o t h e microscope stage d r i v e r through t h e D+7AI/0 b o a r d . F o r each TTL p u l s e r e c e i v e d t h e stage d r i v e r v a r i e s t h e c u r r e n t t o t h e f o u r windings o f each motor i n t h e a p p r o p r i a t e sequence t o r o t a t e t h e motor one s t e p , moving t h e stage 10 um. The stage m i c r o s w i t c h e s p r e v e n t stage o v e r r u n by s i g n a l l i n g t h e computer when they a r e d e p r e s s e d .  25  stage. motor  The  backlash  s t e p s by  t o the  stepping  motors  which provides  the  in  motor  one  direction.  step  The  motors  inverted  may b e  driven  been  either  of  the  from  the  the  which  directly  logic  used  stepping  stage  have  been  would  like  to  Gabriel  Columbia, The  or  (TTL)  pulse  modules  is  for  t h e t r a n s l a t o r module  from  Lam o f  the  translator through  channels  of  the  help  biomedical  i n the designing  computer  9  acknowledge  a  is  a  group  CROMEMCO  are  digital-to-analog  of  providing  CW a n d CCW m o t i o n  for both  with  an  Superior  operation,  and  and  a  some  the  mounted of  on  to  a  This  conversion,  to  the  the  this  given  TRIUMF,  board.  motors.  the  pulses  Ivan  University this  CROMEMCO board seven  two  microscope  interface.  by  the c i r c u i t r y f o r  interfaced  that  o s c i l l a t o r s and t o p r e v e n t  I/O  are  or  supplies,  of  port  (CCW)  microprocessor. in  c o n v e r s i o n a n d one p a r a l l e l i n p u t - o u t p u t  parallel  move  the  freely of  a  analog-to-digital the  power  from  interfaced  D+7AI/0  ampere  We h a v e d e s i g n e d  input  schematic  so  and w i r i n g o f  modules  power  switches  Figure  translator  modules  Installation,  simultaneous  reached.  2.2  described  prevent  limit  screws  o s c i l l a t o r so  translator  modules,  the  lead  two  counterclockwise  own i n t e r n a l  translator  when  about  STM-103  24 v o l t ,  (CW)  the  "Instructions  to  computer and e i t h e r o f from  with  translator  the  to  i n the c o r r e c t sequence t o  its  logic  SLOSYN  S L O - S Y N T r a n s l a t o r M o d u l e T y p e STM 1 0 3 " .  houses  additional  has  a  Hammond  clockwise  module  manual,  improved  nuts.  by  f o u r motor w i n d i n g s  driven  Company  Maintenance of  Dr.  since  each  transistor-transistor  Electric  motors  are  translator  operation  box  has  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  current  the  The  stage  r e p l a c i n g the  s u p p l i e s to each of  the  the  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  The module  of  Liu  of  I and  British  box. SYSTEM-THREE  provides  seven  channels port.  Four  translator  of bits  modules  26  Figure  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 + 7 A I / 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 + 7 A I / 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 a n d 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 modules c o n v e r t e a c h TTL p u l s e s e n t from t h e b o a r d i n t o a sequence o f p u l s e s which p r o v i d e c u r r e n t t o each o f the f o u r motor windings 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 o n e s t e p . The m i c r o s w i t c h e s prevent stage overrun.  27  A  CROMEMCO  JS-1 joystick  D+7AI/0 b o a r d . speaker.  interfaced  to  stage.  i s used  The f o u r  to  control  the input  the observer into  of  buttons  are used f o r  for input  is  required  o p e r a t e d remote a  location classifications.  warm  room.  to  the t r a n s l a t o r  connect  ALDAS  from the computer, The m a n u f a c t u r e r ,  components a r e summarized i n t a b l e 2.4.2  ALDAS s o f t w a r e .  and t h e s t a g e motors driver  to  the  input  through  of  CROMEMCO  the  and a  the  computer  to  are a l s o used signal  The j o y s t i c k i s In  the  alternatives  The s p e a k e r i s u s e d t o  modules.  to  of  These b u t t o n s  from the j o y s t i c k c o n s o l e .  the box which houses  cable  computer  t h e speed and d i r e c t i o n o f  provide branching information to the software. for  the  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 ,  The j o y s t i c k  microscope  is  this  plugged  way o n l y can  be  f o r example i n a l a m i n a r f l o w hood o r  in  specifications,  and  and  ALDAS  one  cost  of  the  system  1.  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 ,  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 D+7AI/0  board  of  the  CROMEMCO  stage  computer.  The  s o f t w a r e communicates 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 sending o r r e c e i v i n g bytes  to  consists  the of  appropriate  seven  port  channels  of  address 8-bit  on  the  D+7AI/0  A/D conversion  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 . d i g i t a l p o r t are a c c e s s e d as i n p u t Figure  10  is  a  list  u t i l i z e d by ALDAS. all  the  and Y a x i s  respectively,  of  the  system  ports  input  of  the  D+7AI/0  board  and s p e a k e r on t h e j o y s t i c k b o x The d i g i t a l  signal  from  the analog s i g n a l s from the  through  analog  port  one  and two  and t h e analog s i g n a l t o t h e speaker i s output through  p o r t one o f t h e D+7AI/0 b o a r d .  of  system.  computer  are  channels  and o u t p u t p o r t s o f t h e computer  through the p a r a l l e l p o r t , joystick  board  and the  The j o y s t i c k , b u t t o n s ,  i s input  and seven  The  These a n a l o g p o r t s  communicate d i r e c t l y w i t h t h e D+7AI/0 b o a r d .  the buttons X  of  board.  The X a x i s o f t h e j o y s t i c k i s  defined  analog  28  TABLE TABLE I :  List  The h a r d w a r e  o f t h e components  components  specifications,  I  o f ALDAS.  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  and approximate  cost i n Canadian d o l l a r s  manufacturer,  a t t h e time o f  purchase, 1982.  COMPONENT  MANUFACTURER  microscope  ZEISS  Model  mechanical stage  SEMPREX  2 mm/rev. o f screw, 7 cm x 12 cm t r a v e l .  $3000  stepping motors  Applied Motion Products  1 2 V , 0 . 1 6 Amp p e r w i n d i n g , 1.8° p e r s t e p  $300  power s u p p l i e s  HAMMOND  2.4 V o l t ,  $92 e a .  motor  SLO SYN  Model STM-103. Up t o 3000 s t e p s / s e c . Has i n t e r n a l o s c i l l a t o r . Accept TTL p u l s e f o r motor  translators  SPECIFICATIONS  COST (Canadian)  ICM-405  2 . 2 Amp.  $240 e a .  step.  ALDAS i n t e r f a c e  CROMEMCO  Model 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  joy  CROMEMCO  2-D v a r i a b l e p o t . 0-5 V o l t s , 4 switches, 0-5V. Speaker.  $235  CROMEMCO (or e q u i v a l e n t )  48 K w o r k i n g s p a c e . 2-8" floppy disks, Z - 8 0 C P U . 4 MHz o p e r a t i o n .  $3000  stick  computer  29  D+7AI/0 p o r t P a r a l l e l port.  Analog p o r t 1  Analog p o r t 2  F i g u r e 10:  Assignment  Comment  b i t s 0-3 a r e t h e o u t p u t o f b u t t o n s 1-4 on t h e j o y s t i c k console.  b i t b-l=0 when b u t t o n b i s depressed. Otherwise b i t b-l=l.  b i t s 4-7 a r e t h e output o f the m i c r o s w i t c h e s which p r e v e n t stage o v e r r u n .  b i t = 0 when c o r r e s p o n d i n g microswitch i s depressed i n d i c a t i n g stage o v e r r u n .  bit  0 i s the input t o the X motor, c l o c k w i s e .  one i n v e r t e d TTL p u l s e p e r motor s t e p .  bit  1 i s the input to the X motor, c o u n t e r clockwise.  one i n v e r t e d TTL p u l s e p e r motor s t e p .  bit  2 i s the input t o the Y motor, c l o c k w i s e .  one i n v e r t e d TTL p u l s e p e r motor s t e p .  b i t 3 i s the input t o the Y motor, c o u n t e r clockwise.  one i n v e r t e d TTL p u t s e p e r motor s t e p .  i n p u t from t h e X a x i s o f the j o y s t i c k .  I/O i s an i n t e g e r from -128 t o 127.  output t o the j o y s t i c k speaker, t  I/O i s an i n t e g e r from -128 t o 127.  i n p u t from t h e Y a x i s o f the j o y s t i c k .  I/O i s an i n t e g e r from -128 t o 127.  I/O PORTS ON THE ANALOG TO DIGITAL CONVERTER.  The CROMEMCO D+7AI/0 board i s i n t e r f a c e d t o t h e CROMEMCO computer ( f i g u r e 8) . The b o a r d h a s one p a r a l l e l I/O ( i n p u t / o u t p u t ) p o r t and seven A/D (analog t o d i g i t a l ) and seven D/A c o n v e r t e r s . Only the d i g i t a l p o r t , two o f t h e A/D p o r t s and one o f t h e D/A p o r t s i s u t i l i z e d by ALDAS.  30  to  be  increasing  positive  positive  from f r o n t  The  stepping  to  stage  supply  each motor  the  correct  draw  160 mA f r o m  ports the  motor  counter  of  the  logic  pulses  power  to  supply  change  clockwise  of  the  three  Fortran,  programming warrant  and  i t s use.  programs.  of  the  joystick. (0-64)  The  each of  resulting buttons  The  value  information  used  axis  increasing  of  0-3  the of  the  t o the main  return  convert  the  the power  a TTL p u l s e  The m o t o r  clockwise  (CW),  the  (CW),  clockwise  are  port.  are  windings  f o r moving the X motor Y motor  There  into  input  interfaced  The  driver  and the  to  is  bits  1 (2.0  Y 0-3  triggered  to l o g i c l e v e l  movement speed  of  joystick computer the  from  guide  LDA  was  by  to  a  5.25  MSCAN,  microscope defines  system  and  utilize  subroutine,  input  assembler,  I/O  subroutine  the  port  read  stage  two  24  in to  these major  provides  for  through  the  number  again.  and  fast for  Of and  ease  sufficiently  programs  the  ALDAS.  has been w r i t t e n  joystick is the  Z-80  for  versatility  version  user's  developed  BASIC,  greatest  The  routine.  programs  available,  compiled  step before  to  through  four  the  stage and  to  board  a TTL p u l s e .  1982).  direction  i n a motor are  the  manual  bit  Y  contains  windings.  (0 t o 0 . 4 V o l t s DC)  A comprehensive  (Faddegon,  subroutines. control  and  driver  when s w i t c h e d o n .  parallel  offered  I/O  the  D+7AI/0  stage  These  languages  latter  the  motor  interactive Fortran  programming the  with  the  (CCW),  t h e t r a i l i n g edge o f  LDA i s a s e t o f  and  necessary  (CCW).  D+7AI/0  from l o g i c l e v e l 0  V o l t s DC),  right  microscope  and t h e  counterclockwise  respectively  the  The  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  X motor  to  communicate  driver.  sequence  left  back.  motors  microscope for  from  is  set The  provide  of to  times zero  joystick branching  31  The of  the  the  automatic  speed  of  s t a g e movement  the  joystick input  microscope  defines  next pulse i s output. the  software.  returns  from  from the  the  variables  required. joystick  2.4.3  Figure  stage  has The  a clock  its  The  control value  runs before  destination,  joystick buttons  branching  the BEEP  subroutines. was  used  Subroutines  cell  joystick.  time that  reached  to  PLINE  and  from the F o r t r a n programs  The  the  for  may b e  information  to  used  locating  program  Three signal  the  OUT50  were  to  return  routine.  along  subroutines  operator used  to  the  program  the main  other  of  through  the  MSCAN a n d ASCAN i s l i s t e d i n t h e a p p e n d i x in  Subroutine  interactively  cells.  used  speaker.  through  length of  and p r o v i d e  The FORTRAN c o d i n g o f the  the  subroutine.  subroutine  stage  ASCAN, p r o v i d e s  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  Once the  subroutine,  with were  through print  the files  LDALOC a n d L D A C L A S S . LDALOC.  LDALOC  is  used  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.  the program the user i s provided w i t h the  following set of  to On  locate entering  options:  LOCATION SCAN MENU:  The  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 . u s e r s u p p l i e d o r computer g e n e r a t e d ) .  B:  Start discrete point scan. (Stage moved between computer generated scan p o i n t s ) .  C:  Define  D:  Redefine  E:  Submit p r i n t o u t  Z:  Exit  location  reference point,  at  maximum  and s c a n p o i n t s  if  are  speed  any.  scan parameters. to  printer.  routine.  scan  Option Z returns  scan boundaries,  Scan p o i n t s  menu  options  t h e u s e r t o CDOS,  are  entered  from  the  computer  t h e CROMEMCO d i s k o p e r a t i n g  console.  system.  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 recordu-rre/it st&je Loca~-trc.cn.. ^Centre celL  Use. the jot/slick "ho control, 'ike. speed,  Ln field  of vieoJ.  .orx,pu.ie.r oudo'X'O-ilcailtj •the staje. o,~t top speed. ficx.t scan, point..  of the sta-cje. dohiue It moves tothe. n&Yjb  /naves 60 -the  SCa.fi poln~t'  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.  Ii  He  generate  contpaier  iXe scon.poCn.xs\ 'NO  (dse the. Joystick £oca.ie. Jznler -ike neu> value, of tne paro.rn.eter.  ts.  Figure 11: LDALOC FLOWCHART. LDALOC i s the location scan program f o r the automated low dose assay system ALDAS. This figure i s a 'bare bones' flowchart of the possible uses of t h i s 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, c e l l s can be located and t h e i r locations recorded by branching to either option A or option B.  33  O p t i o n A: to  record  position  these  c a n be  is  in  computer to the  provided  joystick  option  scan p o i n t s .  s t a g e moves and  A and B a r e u s e d t o  locations.  defined  between the C or  Options  user  C.  The  These  positions  Refer  following  equivalent  to  option  location  or  locations  decrements  the  recorded  to  deleting  the  last  This  stored  then  out  alternative  c l a s s i f i c a t i o n of  "Filename.LDA",  joystick  at  the  the  C for  to  be  and  reference  stage  to  details.  controls the  alternatives  3  classification.  between  the v e s s e l  move option  As  the  speed of  the  scan  entered  from  the  NLOC,  which  is  buttons:  Alternative  prints  stage  causes  Automatic Scan 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 - Delete l a s t recorded 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  computer  in  s u p p l i e d by t h e u s e r t h r o u g h  scan p o i n t the user  the  the  program then  c a n be  generated.  next  with  The  locate cells  in  where returns  memory  Filename  is  the  and  scan enter  useful  Scan  Menu.  to  stage  if  an  the  to  user  can  one  the  following  disk  and  alternatives  classification.  made the  to  reference 1  with in  the  2  the  position stop  stage  from  the  its the  recorded  "LDAPRINT.OUT".  reposition  buttons:  was  4 outputs  and  the  along  error  floppy  Alternatives  The  Manual Scan A l t e r n a t i v e s : 1 - Record l o c a t i o n . 2 - Record l o c a t i o n w i t h 3 - Coincidence check. 4 - Resume s c a n .  location  supplied,  points. of  counter  Alternative  user  microscope  location  recorded  a cell.  computer  the- Location  the  is  exit.  the with  files The and scan the  joystick  34  Alternatives current to  1 and  stage  record  a  Alternative  location  case, manual the  computer  region  location  of  the  If  is  scan  location  counter,  memory.  Alternative  cells  current  recorded  stage  stage and  The  user  at  NLOC,  and  record  2 allows  the  user  is  (See o p t i o n  provided  4 causes a resumption of  is  provided  with  within  D of  the  In  any  with  the  locations.  again  user  location.  recorded l o c a t i o n s are  location.  the  the  recorded  i s moved t o t h o s e  the  Alternative  point.  found  any p r e v i o u s l y  so, the  scan a l t e r n a t i v e s .  next  the  3 c h e c k s t o see i f  Scan Menu.)  no  in  the  c l a s s i f i c a t i o n of  a user defined Location  2 increment  the scan t o  the  automatic  scan  computer  generated.  The  alternatives. Option  B:  The  scan p o i n t s  for  option  B are  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 is  stopped every  through  option  time D.  it  h a s moved a f i x e d  The  user  is  then  interval  provided  with  in turn.  as d e f i n e d the  The  stage  by t h e  following  user  set  of  alternatives:  D i s c r e t e P o i n t Scan A l t e r n a t i v e s : 1 - Record l o c a t i o n . 2 - Go t o m a n u a 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  Alternative stage  is  provided the  user  manual 3  1 records  t h e n moved with to  the  the  to  the  location)  the  reposition  stage  to  and p r o v i d e s  Alternative  next  the  4 outputs  the the the  stage  location  in  computer  memory.  l o c a t i o n a t maximum  speed and t h e  scan a l t e r n a t i v e s .  Alternative  discrete point  scan a l t e r n a t i v e s .  moves  current  exit.  stage  with  the  joystick  and  enter  location  user w i t h the recorded  (without  recording  discrete point  locations  stored  in  scan  2  one  T h e s e a r e t h e same a s f o r o p t i o n A . next  user  The is  allows of  the  Alternative the  current  alternatives.  computer  memory  to  35  files  "Filename.LDA" Option  C:  and "LDAPRINT.OUT"  Option  C  is  used  on t h e  to  floppy  define  disk.  the  following  parameters:  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  the  software.  classification location  2 . The  scans  scan.  If  reference  will  no  begin.  vessel file  (microtest  parameter  defined  flask,  on  A  scan p o i n t s files  are  (n = 0, O p t i o n D:  the  using output  D.  where  from o p t i o n  These are  C:  COINCIDENCE CHECK  0 - 1  D:  COINCIDENCE RANGE  0 -  C.  127  10 -  1270  F:  POINT SPACING  10 -  1270  G:  DATA F I L E  0 - 9  both automatic  (ASCAN)  of  (if  location any)  The  data  is  a  file  is  rewritten  user  and/or  user  is  the  will  be  each type  of  output  supplied  may h a v e  and  for  scan p o i n t s  n  The  list  SCAN PATTERN PARAMETERS E: L I N E SPACING  the  a grid  the  a  program  every  time  up  ten  to  to  the such  scan parameters.  redefine  the user  definable  l i s t e d below:  RANGE 0 - 1 0 10 - 400  A controls  points  vessels or different  PARAMETER A: SPEED B: C E L L COUNT  Parameter  which  scanned.  This  Option D i s used to  program parameters.  scan  input  petri)  disk,  option  for different  9)  are  from  O p t i o n C need o n l y be used once f o r  plate,  "LDALOCn.DAT"  3 . The  scan p o i n t s  p r o v i d e d by t h e s o f t w a r e .  position  maximum  and manual  COMMENT M o t o r s p e e d , 10 = maximum Max. n o . o f l o c a t i o n s t o be recorded. 0 = Automatic c o i n c i d e n c e check 1 = C o i n c i d e n c e c h e c k f r o m MANUAL SCAN ALTERNATIVES Length of square r e g i o n i n which ' to search for coincidences.  speed (MSCAN)  Number o f s t e p s b e t w e e n s c a n lines. Number o f s t e p s b e t w e e n s c a n points, d i s c r e t e point scan. A l s o d i s t a n c e between Y scan b o u n d a r i e s and the computer generated scan p o i n t s . 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".  at  which  the  s c a n modes.  motors  will  turn  Too h i g h a v a l u e  in can  36  result stage  in  missed  i s moving  Parameter  be  of  between  defines  the the  the  when  defines  the  the  in  current  Option the  Parameter file,  region  E:  along  performed  during  with  100 t h e  steps  are  E defines software  software  the  stage  the  seventh  which  classify  The c e l l  cells.  This their  file  i s provided w i t h the  Parameter will D  steps  to  of  store  steps  Parameter  Parameter  n,  a  wide  nearest  discrete  D be  has  number o f  and t h e  the  used  option  submit  the data i n  contains  a  classifications for  computer E the  session  after  contents  all  of  last  of the time  "LDAPRINT.OUT"  LDACLASS.  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  f o l l o w i n g set of  file  listing  the  of  c l a s s i f i c a t i o n program,  Figure  scan  100  the  character,  is  location  generated.  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 2.4.4  by  generated.  during  scan  parameter  long  use.  the  locations  If  the  normal  recorded.  Y scan boundaries  E i s used to  the  using  move  of  if  scan  F  also  point  scan  the the  filename reference  C.  printer.  locations  the are  during  recorded  recorded  100  scan or  be  been  Parameter  "LDALOCn.DAT",  Option  is  the  for  location.  scan p o i n t s  G defines  from o p t i o n  have  of  recommended  B has a v a l u e  current  location.  to  8 is  previously  the  points  interval  line  After  scan  edges  locations to  which  with  the  locations  d i s t a n c e between  data  information  used.  100  coincidence  the  (option B).  to  after  at  A value of  parameter  s c a n l i n e s when t h e  points  of  If  coincident  50,  load.  number o f  B.  region  centered at  F  A or  the  considered value  the  terminated  defines  particularly  a heavy  B limits  using options will  steps,  options:  "LDAPRINT.OUT" the  recorded  location option will  be  scans E  was  erased  location scans. LDACLASS i s u s e d LDACLASS.  The  to  user  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..  Compu.ie.r re.-6re.fvti iAt. cCassc/tco-icon olojia. Jrom. ~/ • <y>/?' o erca.i{re. &6t and. C  r  /  o-p 6cmel 6Ao\-6 &o\ck. c~C.txtsi-Kca,£i.on. occurs.  START  FINISH Com. f>a- 6cr  Compa.ie.r prenyl oa.-6 £/e. tverU conf 6c.-6eol c/t SCSSCen..  £Acs  L^iS 6&  avACCa-ble. a./>ol cttr-re.n.6  £/&  r-tx/iye-Sj \s<z-6ues.  Frxtzr  Ike.  p<xro-m t--6c r •  Figure 1 2 :  LDACLASS FLOWCHART.  LDACLASS i s the c l a s s i f i c a t i o n scan program f o r the automated low dose assay system, ALDAS. This figure i s a 'bare bones' flowchart of the various uses of t h i s program from a user's point of view. Ovals represent branch points and squares show the procedure followed by the computer or the experimenter a f t e r the program branches. For example, c e l l s can be c l a s s i f i e d and t h e i r c l a s s i f i c a t i o n s recorded by branching to option A.  38  CLASSIFICATION  SCAN MENU:  A:  Start  C: D: E: Z:  C a l c u l a t e frequency of c l a s s i f i c a t i o n . Redefine scan parameters. Submit p r i n t o u t t o p r i n t e r . Exit routine.  scan.  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 Option  Z returns  Option  A:  are entered  from the computer  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  Option  to  A  is  used  classify  cells  program then from  the  causes floppy  the  stage  disk  file  supplied.  The c o m p u t e r  locations  with  location  is  a  move  in  turn  "Filename.LDA"  c a n be  given  reached  to  constrained  a l t e r n a t i v e s t o be e n t e r e d  user  is  from the  each  where  (See  provided joystick  the  LDALOC.  the v e s s e l .  location  D.)  The  retrieved is  user  to  those  After  each  stage only  option  with  with  "Filename"  t o move t h e  classification.  the  to  system.  located  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  console.  following  set  of  classify  the  buttons:  C l a s s i f i c a t i o n Scan A l t e r n a t i v e s : 1 - Classify. 2 - Move s t a g e . 3 - Resume s c a n . 4 - Exit routine.  If  alternative  colony  at  joystick by  the  each  s e l e c t e d the  location.  buttons. user,  classification their  1 is  If  of  5.  a location is  is  The  complete.  an o p p o r t u n i t y  are Up t o  reached  scan  computer  which  has  c l a s s i f i c a t i o n of  (classification  classifications  classification  A  user  output  a file to  sets of  is  entered  LDACLASS b u t  alternative  maintains  five  in  1-4  to  3) of  it cell  not is  using  classified given  locations  "Filename.LDA" classifications  the  when  a and the  (including  39  the  classification in  per c e l l  location.  Classification 2 - 5  (default  may is  reclassify  first  any  classification within  c l a s s i f i c a t i o n set  set equal zero).  If  stage  joystick  with  the  alternative  for  2 is  and  subsequently  resume  coordinates  not  the computer t u r n s C:  classification ("Filename" set  of  Option  frequency  a given  of  The  the  classification  the  number  one c a n d e t e r m i n e  the  that  s u r v i v e d and t h e  computer  can be  were  killed  of  occurences  as  the  number t h a t w e r e k i l l e d b y  of  scan is,  or  each  is calculated for include  if  2-'sick  c l a s s i f i c a t i o n scan  well  a  "Filename.LDA".  example,  t h e number o f h e a l t h y as  that  c l a s s i f i c a t i o n set  For  the  make  l a t t e r case the  constrained to  a given  calculation.  the  that  stage.  A separate frequency  a r e c l a s s i f i e d as 1 - ' h e a l t h y '  c l a s s i f i e d during  scan,  s t a g e i s moved b y t h e u s e r ;  classification in  2-'killed', number  computes  c l a s s i f i c a t i o n set  i n each c l a s s i f i c a t i o n set i n the data f i l e  l o c a t i o n scan c e l l s are  C  i s user supplied).  of  In t h e  a b l i n d eye t o t h e m o t i o n o f t h e  classifications.  locations  cells  a d j u s t e d when t h e  any  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  or reset the scan coordinates.  Option  allowed  which a l l c l a s s i f i c a t i o n s i n  classification, are  are  Classification Definition 0 Location never reached during c l a s s i f i cation scan. 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 Location reached during 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 .  Sets  1 - 5  user  c l a s s i f i c a t i o n s e t one)  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 a s f o l l o w s :  1 - 5  The  LDALOC, w h i c h i s  as  cells  number  of  in  during  each only the the  and l a t e r  the  1-'survivor'  or  1  that sick  s e t t i n g parameter  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 prompt the computer f o r t h e  survived cells F to  and that  1 using  results.  40  Option  D:  Option D i s used to l i s t  program parameters.  and/or  RANGE 0 - 1 0 0 - 2  SET TO C L A S S I F Y  PARAMETERS  0 = 1-5 = E: C L A S S I F I C A T I O N VALUE 0 - 5 Class C L A S S I F I C A T I O N FREQUENCY CONSTRAINT PARAMETERS F: C L A S S I F I C A T I O N SET 0 - 5 0 = 1-5 =  only be  •healthy'  a n d C n e e d no e x p l a n a t i o n .  for  when  healthy  cells  LDALOC)  and  can  0  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 scanned;  be  given  example,  to  E  to  classify  classification  were  "LDALOC"  2.  cells  set,  or  the  D to  No c o n s t r a i n t Set c o n t a i n i n g  1,  frequency  classification Option to  the  E:  of  (the  E is  classified  This  file  printer.  t h r o u g h o p t i o n D.  used to  location  contains  a  printout.  2-  constraint  locations  calculation  the  listing  scan  the  of  is  desired  data i n of  the  in  parameters  a  in  for  a  given  Parameter F i s used i f  A f t e r u s i n g o p t i o n D the contents of  b e e r a s e d a n d r e p l a c e d b y new  or  (classification =0)  classify  submit  'sick'  c l a s s i f i c a t i o n set used  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 Option  to  selectively  not  classification  are  user  could  1-  if  value  as  c l a s s i f i c a t i o n within a given c l a s s i f i c a t i o n set. separate  a given  classified  colony  only  constraint  P a r a m e t e r s D and E a r e u s e d  The  to  No c o n s t r a i n t Set c o n t a i n i n g c o n s t r a n t . o f l o c a t i o n s t o be s c a n n e d .  classified with  cells  s e t t i n g parameter  parameter  used  if  located with  by  definable  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 frequencies only. 2 = A l l printout 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 cations .  0 - 6  COLONY LOCATION CONSTRAINT D: C L A S S I F I C A T I O N SET  B,  the user  These are l i s t e d b e l o w :  PARAMETER A: SPEED B: PRINTOUT  Parameters A,  redefine  a  each  previously.  file  "LDAPRINT.OUT"  printout  requested  "LDAPRINT.OUT"  will  41  PURPOSE AND  3.1  PURPOSE OF THE The  assay'  purpose  which  is  EXPERIMENTS.  of  these  used  to  experiments  measure  cell  t h a n 50%, b y r e d u c i n g e x p e r i m e n t e r increasing  productivity.  s u r v i v a l of ranging  concentrations model of  cell  are able (about at  line. the the  is  definition  dose  of  is  'low  dose  from  the  effect  cells  of  the  survival  levels  of  parameters  effect  of  the  is  the  Survival  is  relative  generally  radiation,  to  measure  the  assay'  is  used  0.50-0.90  in  with  (J.Brosing  varying  thesis,  to  greater  for  of  1983).  reduce experimental survival  of  the  error  low dose  and t o  mammalian  clinical  assay  reduce  implications.  at  low doses  Previous  of  than  0.50  with  consequently  of  of  survival ±1%-±10% is  this  experimenter  s p e e d up t h e e x p e r i m e n t a l cells  cell  assay  to  improving  survival the  order  is  to  the  days  of  1956;  This  which  seven  measuring  the  The  cells  in  efficiency  laboratory  cell.  of  normalized  levels  uncertainties  a  cells  Puck and M a r c u s ,  our  PhD  number  fifty  plating  survival  a s s a y as d e s c r i b e d by  about  important  the  radiation  of  capacity of  A major p o r t i o n  has  on  and  linear-quadratic  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 .  The  dose  greater  misonidazole  the  proliferative  survival  ionizing  deviation.  devoted  'low  exposed to doses of  determine  on J t h e  the  reducing experimental error,  f o r m c o l o n i e s o f more t h a n  difficult  conventional  standard  to  of  generations).  It  levels  a measure  to divide  14  zero  misonidazole  To  at  improve  survival.  Survival operational  (CHO)  3.  1 . To  survival  2 . To m e a s u r e  1.5-30 Gray. of  is:  strain,  Chinese hamster ovary  from  PROCEDURE  time thesis  strain,  procedure.  ionizing  investigators  irradiation have  studied  42  the  effects  mammalian  of  cells  (Adams e t  al,  generally  been  ( 0 . 1 - 5 0 mM) indicate  for  not  1976;  cell  (e.g.  experiments  Moore  damaging  the  tumours  which  findings  OER o f  CHO c e l l s  et  by  oxic  this  may w e l l  the  of  would  several i n the  weeks. clinic.  on t h e  on t h e  at  fractions  of  s u r v i v a l of  less  of  of  high  (figure of  results  under  anoxic is  anoxic  clinical  than  about  1973;  cells In  a n o x i c CHO c e l l s  without  2).  oxygen Wardman,  are  each  over  generally  as  1977)  If  this  at  clinical  thesis  subjected to  the  doses effect  measured  analogous  the  less  of  270 k V P X - r a y s  to  treatment course  than  of  0 . 1 mM has,  radiation  of  the  act  DMF  levels),  of  literature  A t y p i c a l tumour  2 Gy  when  environment.  the misonidazole survival  these  advantage  Other  and  oxic  of  cells  anoxic  2 Gy.  does  under  conditions  an  about  Misonidazole  t o measure what e f f e c t m i s o n i d a z o l e  mammalian  misonidazole.  of  in  (Adams,  concentrations  i s important  survival  concentrations  20  Misonidazole It  of  a reduction  the oxygen e f f e c t  involve  kill  misonidazole  (i.e.,  The  extent  the  have  misonidazole  show a s i g n i f i c a n t r e d u c t i o n  a s i m i l a r mechanism. be  under  are  of  conditions  15 mM m i s o n i d a z o l e  measureable  the  (1982)  of  Gy).  CHO c e l l s .  which  doses  (5-30  important  cells  al to  of  Thus  an  that  irradiation  reduction of  today  any  so t h e r e  low doses  et  hypothesis by  any  increase  anoxic  radiosensitizer  asynchronous  1976)  survival  Such i n v e s t i g a t i o n s  radiation  (DMF)  to  the  concentrations  good  cells,  irradiated  the  a  al,  contain  Palcic  of  on  under  1976).  high  factor  of  will  radiosensitizers  at  is  survival  misonidazole  Recent  is  doses  misonidazole  the  conditions.  supports  high  radiation  McNally,  fairly  The d o s e m o d i f y i n g  effect  treating  at  suspension  misonidazole  ionizing  1976;  out  fairly  that  to  Moore,  carried  a dilute  further  radiosensitizer  subjected  and  conditions. 2.5  the  if and  misonidazole is  measured  43  over  a wide range  (0-30  Gray) .  of  sensitizer concentration  Cells  misonidazole  were  i n order  kept  on  to minimize  The s u r v i v a l o f m a m m a l i a n important of  the  radiobiological  cell  survival  measurements multitarget  at  theory  linear-quadratic and  Leenhouts,  Because number  of of  history,  the  etc,  impossible)  to  minimum  experimental parameters general our  these  In order  development independent  of  of  1962).  and  to the  ( 0 - 5 Gy)  based  models  was  Experiment  set  s u r v i v a l on m i s o n i d a z o l e  Chadwick  is on  difficult the  to  (perhaps  basis  of  cell  a model i s t o  describe of  or  to  1980).  cell  changes  models  a  for  use  set in  of  basic  contribute  to  our  direction  distinguish  contribution how  in  between  these models  determine  to  the  environment,  vary with varying misonidazole performed  include  1962) ,  1978;  on  cell  My to  shape  large  and p r o v i d e  verification.  were  the  (C.Tobias,  p r e d i c t i o n s made b y  models  predict  the  effect  a s u i t a b l e model  has  (due  it  way  radiation  data  adequately  study  survival.  with  treatment  Gooch,  The g o a l o f  of  field  as  models  the  experiments  on c e l l  t h e dependence  these  this  radiobiological  of  such  In  experimental  of  Such  model  biological  survival)  predict  e x i s t i n g models,  parameters  sets  misonidazole of  to  cell  between  find  Gy).  Bender  systems.  to  dose  K e l l e r e r and R o s s i ,  in  hypotheses  of  radiation  (5-30  parameters  to  and r a d i a t i o n  ionizing  models  repair-misrepair  effect  and  understanding  subjected  1966;  Bender and Gooch,  systems  research.  Two  can  of  1956;  uncertainty  acceptable  within  dose  radiation  the  distinguish  the p r a c t i c a l i t y of be  and  Survival  Marcus,  correlated  (e.g.  of  and  time  at low doses of  low  (Sinclair,  inherent  which  s u r v i v a l data a  (Puck  1981),  highly  cells  of  the  mM)  cytotoxic effects.  at  doses  model  during  implications.  curve  high  ice  (0-40  must  to  the  some  dose  concentration.  measure  A showed t h e  the  effect  of  general  trend  c o n c e n t r a t i o n up t o t h e  level  44  at  which  data  no f u r t h e r  from  increase  experiment  proportional  in cell  kill  set A indicated  to the X-ray  dose  i s observed.  that  the log of  and t o t h e square  of  Analysis  of the  t h e s u r v i v a l was the X-ray  dose  as  follows:  2 log^Q S = -aD-bD  Equation 3  S=cell survival. D=X-ray dose. a=alpha, a m i s o n i d a z o l e dependent parameter. b=beta, a m i s o n i d a z o l e dependent parameter.  The  choice of equation  toward  the molecular  3 for fitting  theory  of cell  the data survival  consistent with t h i s survival equation. the  simplest which can adequately  as an i n f i n i t e data  reported  sufficient For dose,  polynomial in  the  alpha,  remained  constant  quadratic  component  0.0023 Gray of  -2  to  linear  s e t B.  0.042±0.010 G r a y  of  the  This  -2  in  set of  uncertainty  misonidazole  concentration  experimental  dose,  l o w dose  statistics  and range  assay  .  o f doses  The  of  carefully  (ALDAS)  are  of the  concentration but  from  of  range  the motivation f o r  designed define  was used  the  approximately  concentration  results provided was  terms  component  to  alpha  i n w h i c h b e t a was c h a n g i n g system  experimental  coefficient  the misonidazole  more  which i s  used.  increased  experiments  to  a bias  c a n be w r i t t e n  and q u a d r a t i c  of the linear  1  beta,  These p r e l i m i n a r y  experimental  automated  majority  s i g n i f i c a n t l y with misonidazole  0.0065 Gray  O . l m M t o 4 0 . 0 mM.  experiment  The  the  set A the coefficient  at  model  A l l models  For the great  literature  d i d not vary  o r any o t h e r  f i t the data.  i n dose.  not constitute  T h i s e q u a t i o n was chosen a s one o f  t o f i t s u r v i v a l d a t a over t h e range  experiment  does  to  most help  minimize in  the  quickly. improve  and reduce experimental u n c e r t a i n t y by a l l o w i n g f o r  45  improved  experimental  experimenter set  and  by  reducing  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 .  B show e q u a t i o n  constant  design  at  3 t o be a reasonable  0.039±0.010 G r a y -2  0.0019±0.0002 G r a y  to  the  strain  The r e s u l t s o f  f i t to the data. and  0.01210.002 Gray  beta -2  in  on  the  experiment  Alpha  remains  increases  from  the  misonidazole  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  was:  Purpose  and procedure.  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  varying  concentrations  exposed  to  X-rays.  misonidazole coefficients  of  misonidazole  2 . To m e a s u r e  concentration. alpha  The p u r p o s e  of this  f o r measuring  on c e l l  survival  t h e dependence  3 . To  s e t of experiments  estimate  the  and b e t a o f t h e s u r v i v a l curve  of  the effects of when  cell  linear  cells  are  survival  and  on  quadratic  described by equation 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  cells  from  spinner  flasks  (suspension  culture)  were  diluted  to  5 1X10  cells/ml  doubling  time  eleven of  hours  prior  approximately  to  irradiation.  14 h o u r s ,  and they  The c e l l s  remained  growing throughout the time course o f the experiment. was p r e p a r e d were  prepared  2.8X10^ minutes serum  i n bulk  cells  by  from which  further  were  a t 600 r . p . m .  removed  the required  dilution from  the  and resuspended  (FCS) a n d no NaHCO^,  with  vortexed,  medium.  then  placed  (N^,  less  than  in irradiation  5 ppm 0 ) o  concentrations irradiation  centrifuged  for six  i n 1 m l a l p h a medium w i t h f e t a l into  added  19 m l o f  as r e q u i r e d .  v e s s e l s on i c e and p u r i f i e d  was f l o w e d  exponentially  Before  suspension,  and p i p e t t e d  into  a  5 - 4 0 mM m i s o n i d a z o l e  misonidazole  medium w i t h F C S a n d n o NaHCO^ w i t h m i s o n i d a z o l e were  have  the v e s s e l over  calf alpha Cells  n i t r o g e n gas  t h e medium f o r  46  a  period  of  45-90  X-ray  machine  using  ferrous  (270  vol.  irradiated  on  one  In  at  exposed  those  1.5-2  plate  hours.  for  the  misonidazole 3.2.2  the  and  flowing of  of  to  An a v e r a g e low  dose  of  the  Choosing between the  conventional 20-30  (before  automation,  requires  about  dishes  suitable  five  with  only  assay  minutes  low dose  when assay.  primarily  the  to  low  minutes  it The  to  the  irradiated  plate,  in  of  the  samples  the  number (if  one  The  for  were and  plated  low  using  The  s t a i n and  upon  six  of  cells  the c e l l s  needs  to  low  capacity  assay  requires  100-200  cells,  conventional  assay  count  three  two  to  assay  assay  plated.  about  is  is  more  of  the  limited  This  are normally  plate  hours. The  uncertainty  conventional  assay  different  assay.  low dose  the  the  microtest  with  classify  the  six  assay,  conventional  dose  to  Cells  low dose  the course of  and  Thus  markedly  accuracy  uncertainty  the  ml  up  misonidazole.  vessels  minutes).  colonies each.  improves  0.5-3.0  were p l a t e d p e r  four  locate  45  were  irradiation  assaying c e l l p r o l i f e r a t i v e  plate,  r e d u c e d b y e i t h e r p l a t i n g more c e l l s throughout  150 c e l l s  doses.  count,  Radiation  Samples  of  low dose and c o n v e n t i o n a l  at  in  centrifuged  remove  the  approximately  100-200  conventional by  to  were  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  approximately  petri  cells  to  Picker  and s t o r e d on i c e f o r  for  Up  a  calculated  the  aliquots  radiation  d o s e a s s a y i s a more s u i t a b l e means o f than  through  of  assay.  was  1966).  were p l a t e d u s i n g  about  was  and H a r t ,  doses  plating  rate  ed,  constantly  plating  higher  Cell  (Fricke  (no FCS)  used  dose  irradiation,  irradiation  the  The  Toesch  experiments  prior  irradiation  techniques Attix  gas  2  of  to  assay.  N  of  Gy/min) .  m l a l p h a medium  some  exposed  167,  course  low doses  conventional took  the 9.5  source  0.40-1.60  page  600 r . p . m .  to  The  dosimetry  ice with  into  hour.  minutes  and  II,  During  were removed  kVP,  sulphate  Dosimetry,  vessel.  minutes.  can  be  distributed  four  times  as  47  many c e l l s  to  reduce  the  counting  the  4.1.3  i s shown t h a t  be  it  number o f  necessary  to  (approximately same  these  cells  plate  as w i t h This  assays.  is  plated  as  dishes  the  low  takes  much  the  100  of  two)  low dose  cells,  survivors  assay  into  easier  in  1500  of  dose  a factor  or  by  accurately  assay.  In  section  50% s u r v i v a l o f p l a t e d c e l l s  approximately  analysis  It  by  i n the case of  seven p e t r i  accuracy  classified.  uncertainty  when  account  to  200 the  plate  and  it  would  survivors  and  each),  to  achieve  the  are  located  and  cells  plating  killed,  efficiencies  subsequently  handle  of one  2 vessel must be  (microtest  plate,  counted,  diluted,  errors) . cells  at  In  practice  levels greater  3.2.3  Survival  we  25cm  1. F o r  for  etc.  use  low  than  data.  the  than  low  cells  classified  total  number  independently dose  assay  dose  as  Survival  data are  assay.  assay  survivors  cells  on  the  is  experiment.  colonies plated,  counted  dose  conventional after  for  the  assay  staining  according to equation dose  by  of  experiment.  i s estimated according to equation was u s e d  to  the  are  is  as  the  handled  number  divided  by  corrected for  The  standard  The 9,  estimated  standard  section  estimate  the  of the  possible to  the  deviation  5.  c a l c u l a t e d as the  the  low  follows:  number  number  The s u r v i v a l i s n o r m a l i z e d  survival  test  the  divided  at  chi-squared  for  1.  survival  survival  The  zero  survival  The s u r v i v a l i s n o r m a l i z e d  s u r v i v a l i s estimated according to equation the  (which  systematic  survival  as  l o c a t i o n day,  at  For  measure  c l a s s i f i c a t i o n day  average  2.  reduce  calculated  2.  the  to  dishes  obtained with either  according to equation  of  to  C a l c u l a t i o n s of  survival  l o c a t e d on t h e  zero  petri  50%.  location errors, survival  ten  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  the  of  etc.)  plated,  dose a s s a y o r t h e c o n v e n t i o n a l differently  flask,  of  to the  deviation  of  cells average of  the  4.1.3.  most  likely  values  of  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 as w e l l as t h e i r results  3.3  70% c o n f i d e n c e i n t e r v a l s .  Motivation  survival  perform  the  levels  this  experiment  for  to  the  cell.  assay.  of  about  to  healthy,  The  3) the  or  is useful  greater.  assay procedure.  for  The  mainly  due  to  eyes on g r a p h  derive  minimum  i s the danger of dividing  equipment  cells  in a large  be  to manually  performed  benefits  Data  about  to  the  thereby  adding  a  for  strain  locate  location  sufficient  speed  conventional  a  of and  survival  a t i r e d experimenter missing c e l l s  and  to  2 mm d i a m e t e r  record the  with  over  needed  The a s s a y i s a  f i e l d of  low  survival  stage.  concentration required  paper  must  statistical  the  The  measuring c e l l  a microscope with a manually driven  procedure  automated  over the manual stage can be  low  dose  assay  stepping-motor-driven  c o n t r o l l e d by t h e  observer  system.  system  in  favour  systematic error  microscope  The c o m p u t e r a higher  r e c o g n i z e d more e a s i l y . experimenter  recording the c e l l  i n which the c e l l s  (ALDAS)  the  objective  power  This  The  results  task of  in  a  stage  ALDAS o f f e r s  takes over  strain.  locations,  c o n s i s t s of  stage.  through a j o y s t i c k .  l o c a t i o n , thus  r e c o g n i t i o n and l e s s task of  section four  low dose  to  the  results.  controlled,  the  improved  10 um d i a m e t e r  the  This  There  survival  an  0.50  assay i s  refocus  accuracy  cells  of  experimenter,  and  of  D e t a i l s o f t h e a n a l y s i s and  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 .  small c e l l  of  (equation  EXPERIMENT SET B .  dose assay d e s c r i b e d i n  on  linear-quadratic equation  are p r e s e n t e d i n s e c t i o n 4.  3.3.1  at  the  computermotion  many  benefits  keeping  c a n be u s e d fewer  is  errors  track  so  that  in  cell  The c o m p u t e r a l s o t a k e s o v e r  thus  a reference grid  a r e p l a t e d i s not r e q u i r e d and c e l l s  on t h e  the  vessel  c a n be p l a t e d i n  25  49  cm  flasks  flasks  instead  classify.  plates. better  The than  closely cells is  microtest  plates.  i s much e a s i e r a n d f a s t e r .  w a l l when p l a t e d and  of  and  Phase  s e t A)  optics  automated 100 um o r still  of  be  easy  density  greatly  cell to  be  Cells plated  identify  effectively  improves  the  so t h a t  on t h e  the low dose assay procedures (experiment  set  advantages  ALDAS  used before  ALDAS  include  speed  and  time,  dose,  concentration. recorder.  more  Plating following  (experiment  accuracy  in  of  to  1.  involving  a function  accuracy  ALDAS  new e x p e r i m e n t s  as  microtest  The  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 morphology  locate  2 C e l l s p l a t e d i n 25cm area flasks. 2. L o c a t e c e l l s i n the 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 . Cells located 2-3 days 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 the computer t o r e c o r d the 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 of 1 0 - 1 0 0 um. 4. 6-8 days f o l l o w i n g i r r a d i a t i o n use the computer 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 each 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 calculates survival.  plates.  of  with  c l a s s i f i c a t i o n day.  procedure After  ALDAS in microtest  to  can be p l a t e d  locate.  in  B):  Locate c e l l s i n the m i c r o t e s t p l a t e s by m a n u a l l y moving t h e microscope stage. Cells located 2-3 days 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 paper. R e p e a t a b i l i t y about 5 0 0 - 1 0 0 0 um. 4. 6-8 days 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 to the approximate 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 classify. 5 . A d d up t h e number o f c e l l s w i t h each g i v e n c l a s s i f i c a t i o n and calculate survival.  cell  cells  contact a well  location  cells  a l s o makes t h e m e a s i e r t o  2.  Additional  used  diameters  Low Dose A s 1.  locating  C e l l s a r e more l i k e l y t o  cannot  system  5-10  a n d a f t e r ALDAS  Before  and  i n m i c r o t e s t p l a t e s , m a k i n g t h e m more d i f f i c u l t  a t , a higher  a summary  Plating  The l a t t e r w o u l d i n v o l v e  radiation  the use of  and  the  in  data  changes  sensitizer  a t e l e v i s i o n camera  and  50  3.3.2  ALDAS a n d t h e  was u s e d i n dose  experiment  assay.  (figure  Cells  5).  that  flask  and  the  the  holder.  be  assayed the  was h e l d could  The  screws.  sides  2.  The  opposite  rectangular  on  of  the  were  for  plated  the  flask.  in  entire  were  LOCATION  SCAN  MENU  the  the  The  holder  flask  flask.  (figure  5)  was  were This  C  located defines  was l o c a t e d .  essentially  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  used f o r  samples.  After experiments culture about with  the  the  scan  could  boundaries  go  ahead.  f l a s k s and i n c u b a t e d  1-20 the  cells  help  of  (qualitatively)  and  Irradiated two t o  original  of  the  ALDAS.  In  trial  the  plating  on  the  w h i c h were same  day  so  irradiated that  they  three  the  with could  the all  define  of  the  two  stage  reference  were  identical, this  input. the  position were  were  plated The  cells)  with  defined  in  the  were it  with  then was the  be  concentration c o r r e c t e d by  of the  the  25 c m  2  of  located observed  number  T h u s i t was i m p o r t a n t t o l o c a t e same  were  small colonies  ALDAS  increases  low  boundary  The same c o o r d i n a t e s  plated  efficiency  to  limits  Next  stage  First  order  the  hitting  the  used. in  such  possible  f l a s k s used f o r  days.  experiments  days between i r r a d i a t i o n and l o c a t i o n . samples  cells  for  (clones  that  repeated.  reference  to  No s c a n p o i n t s  set  B were  as  flask without  option  dose  a l l of  rigidly  parallel  Since a l l the  experiment  low  flasks  was d e s i g n e d  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 .  flask  with the  culture  2  oriented  software.  for  as  the  These were p r o v i d e d by t h e assay  cells  and r e f e r e n c e p o i n t o f  area of  flask  of  of  25 c m  specific orientation  the  of  survival  An e m p t y f l a s k was s e t o n t h e s t a g e o n a  scan the  boundary  the  1.  a  corners  movement d u r i n g t h e position  in  T h i s s e c t i o n d e s c r i b e s how ALDAS  scan boundaries  as f o l l o w s :  objective  diagonally the  to  s p e c i f i c a l l y designed  the  lead  s e t B t o measure  Before p l a t i n g ,  f l a s k were d e f i n e d holder  low dose a s s a y .  of all  misonidazole same  plating  51  efficiency  as  measured  C e l l s were l o c a t e d as 1.  The  flask  orientation  as  the  by  the  zero  irradiation  dose  sample.  follows:  was  mounted  flask  used  on  to  the  microscope  define  the  stage  scan boundaries  in  the  same  and  reference  position. 2. The  LDALOC LOCATION SCAN MENU o p t i o n A ,  name o f  the  dataset  to which recorded  'Start regular l o c a t i o n s were  s c a n ' , was u s e d . to  be o u t p u t  was  entered.  the  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  4.  After  automatic  controlled, The  s c a n mode;  was  used  generated  locate the c e l l s 5.  The  automated console  the  that  to  i n the  joystick  was  pressed  by  the  to  automatic  record  the  the  d i r e c t i o n of  control  the  scan  speed  point.  of  The  was  used  If  a cell stop  manual  to  the  control  medium  in  the  each  c o r r e c t pH o f  lid flask  7.4.  the  the  scan  automated  mode  s t a g e was  stage  by  software joystick.  to  the  now  first  began  in  which  of so  the that  scan  speed  button scan. the  one The  to  the  on t h e  to  stage  joystick  and b u t t o n one on t h e The  stage  was  next  joystick was  then  controls  both  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  location. s t e p was  the  was o b s e r v e d ,  the stage.  s c a n and t h i s  37°C w i t h  the  controlled  experimenter  cell  j o y s t i c k c o n s o l e was  then  controlled  by  the  repeated.  A f t e r the c e l l s were l o c a t e d i n each c u l t u r e at  s t a g e was  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  under the microscope c r o s s h a i r s pressed  position  flask.  to  d i r e c t i o n and speed o f  is,  the  automatic  scan p o i n t .  controlled  reference  and the speed o f  joystick  software  locating  joystick.  flask C0  2  tightly flow  f l a s k t h e y were  closed.  was  not  There  needed  was to  incubated sufficient  maintain  the  52  Six  to  incubator  seven days  and t h e  irradiation  LDACLASS C L A S S I F I C A T I O N  2.  The  name o f  the  dataset  3.  The f l a s k r e f e r e n c e  4.  The  s t a g e was moved  at  this  Button  section  2.3  2 for  was moved  repeated.  with  was  A,  'Start  the  from  follows:  s c a n ' , was  locations  the  of  used.  the  cells  entered. was l o c a t e d u s i n g t h e  either  joystick  entered.  a  The  3 -  to  the  first  monitored  complete  all  of  returned  to  console  joystick. cell  location.  through  description to  the the  the  the  reference  pressed  The  microscope  4 - possible  these  cell  were  the  colony  were  used:  error.  (See  classifications.)  location  and t h i s  classified  postition  output  then  classifications  and  of  next  cells  c l a s s i f i c a t i o n s were  was  following  coincidence,  automatically  the  contains  automatically  the  killed,  After  automatically along  of  was  survivor,  removed  TV.  one  classification  stage  which  point  location  e y e p i e c e o r on t h e 5.  f l a s k s were  SCAN MENU o p t i o n  f l a s k t o be c l a s s i f i e d was  colony  the  c e l l s w e r e c l a s s i f i e d u s i n g p r o g r a m LDACLASS a s  1.  i n the  1 -  after  to  the  and  the  the  step  stage  cell  dataset  The was was  locations  named  by  the  was u s e d t o d e t e r m i n e  the  user. After number  of  generally  the  c e l l s were c l a s s i f i e d the  colonies  with  each  no c l a s s i f i c a t i o n was made  classification  per  colony  classification  scan.  Thus  to  given  determine  the  classification. during  (recorded the  computer  default  the  For  location  location)  was  experiment  The  results  one  made  the  were were  follows: 1.  LDACLASS C L A S S I F I C A T I O N  SCAN MENU o p t i o n  B  s c a n and o n l y  program parameters  c l a s s i f i c a t i o n frequency.  set  C was u s e d .  using  sufficient obtained  as  53  2.  The  frequency 3. in  the  name o f  was  dataset  for  which to  The c o m p u t e r o u t p u t t h e number o f dataset.  3.3.3  From t h e measured i n s u r v i v a l were  Purpose  indicated  that  concentration  and  beta in  changed.  misonidazole  determine  0.1,  changed  the  classification  range  most  of  The  results  rapidly  as  0 . 1 - 5 . 0 mM.  It  set  1.5,  and  corresponding  0.6,  0.2,  1.  In  survival  order  prepare a l l  B  was  the  would  be  the  figure  low  to  were  0.01,  dose  and  of  survival  experiment  function  is  not  to  of  set  A  misonidazole  clear  whether  measure  the  for  addition,  the  a  concentration of  irradiated. to  alpha  effect  of  The  first  same  set A w i t h the uncertainty  five  following  due  to  estimate  misonidazole  10 mM m i s o n i d a z o l e  assayed  1.0,  five  procedures  misonidazole  was  l e v e l s of  the  straightforward  all  Survival  survival  0.003,  assay.  experiment  obtain  measured In  mM)  reduce  of  0.85,  for  modifications:  this  solution  between  variation,  concentrations  stock  were  used  variation of  0.7  which  were  at  was  in  each  used  to  samples. would  irradiated  culture  and  flask  experiment.  irradiate  samples.  irradiated  13.  a  designed  approximately  0.03,  to  One p e r s o n  plate  of  and  was  experiment.  spinner  the  5.0  s e t B as f o r  experiments  2.  0.1,  using  experiment  the  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 .  ten doses  assayed  frequencies  occured  estimated.  procedure.  Experiment  0.4,  0.5,  classification  times each c l a s s i f i c a t i o n  F i v e v e s s e l s each c o n t a i n i n g a d i f f e r e n t (0,  the  entered.  and u n c e r t a i n t y  also  the  plated  were  In  the this  within  growing  A t y p i c a l time  samples way  while  all  seven  five  hours.  exponentially course  for  the  another  person  irradiation The  cells  throughout experiment  the is  would  vessels from  the  duration shown  in  54  F i g u r e 13:  TIME COURSE OF EXPERIMENTS SET B.  In o r d e r t o reduce e x p e r i m e n t a l e r r o r f o r experiment set B c e l l samples w i t h 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 were i r r a d i a t e d each experiment and t h e time taken from t h e p r e p a r a t i o n o f each sample o f c e l l s t o t h e p l a t i n g o f t h e c e l l s was kept t h e same. To do t h i s i t was n e c e s s a r y t o a c c u r a t e l y time t h e e x p e r i m e n t a l procedure as shown i n t h i s figure. The numbers r e p r e s e n t t h e following concentrations of misonidazole: I - 0 mM; I I - 0.1 mM; I I I - 0.4 mM; IV - 1.5 mM; V - 5.0 mM.  55  3.  Cells  minutes plated  plated  at  600  r.p.m.  for  the  low dose  diluted  2000 t o  the t o x i c 4.  the  the  prior  conventional  to  plating  a s s a y were  5000 f o l d ,  to  not  assay  were  remove  the  centrifuged  centrifuged  for  misonidazole.  s i n c e the  6  Cells  samples  were  reducing the concentration of misonidazole  below  level.  With previous  s u r v i v a l curves of  for  for  microtest  classification  procedures  each experiment.  plates  of  i t w o u l d be v e r y  the  used  cells  for  The l i m i t i n g f a c t o r s w e r e t h e  the  plated  d i f f i c u l t t o measure  in  low dose  assay  and  the  the m i c r o t e s t p l a t e s .  five  plating  location  and  Cells for  the  2 low dose save  assay  time  in  system  (ALDAS)  plate,  locate,  plated  to  experiment  plating  and  was u s e d  to  classify,  in a flask in  minimum o f due  for  observer  strain  in  cell  B were  compute  the  3.3.4 as The  for  experiment  chi-squared  and b e t a and t h e  data.  set  A.  Table  t e s t was u s e d t o 70%,  95%,  were  low  flasks dose  100  this  to  to  assay  W i t h ALDAS one of  200  can cells  assay would take  qualitatively  less  spaced c e l l s  under  The d e t a i l s o f  the modified  a  reliable a  lower  low dose  3.3.2.  survival 2  area  automated  Previously  in section The  25 cm  survival  l o c a t i n g more w i d e l y  are presented  Survival  the  results  m a g n i f i c a t i o n through the microscope. assay procedure  The  in  classify cells.  30 m i n u t e s .  and  plated  location.  l o c a t e and and  20 t o  45-50 minutes  set  was e s t i m a t e d  summarizes  estimate  the  the most  and 99.9% c o n f i d e n c e  in  the  experiment  same set  l i k e l y values  intervals.  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.  manner B  data.  of  alpha  D e t a i l s of  the  56  TABLE I I Table  II:  THE AVERAGED SURVIVAL DATA OF EXPERIMENT SET B .  The s u r v i v a l a s m e a s u r e d b y t h e l o w d o s e a s s a y (LD) a n d t h e c o n v e n t i o n a l a s s a y (CV) w a s 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  0 mM M i s o n i d a z o l e : 0 LD/CV 1.00 161 0.75 LD 236 0.85 LD 310 0.72 LD 470 0.68 LD 631 0.56 LD 814 L D / C V 0.37 1500 0.18 CV 1920 0.089 CV 2290 0.033 CV 2710 0.0066 CV 3010 0.0022 CV  0.00 0.20 0.09 0.20 0.08 0.06 0.06 0.13 0.057 0.009 0.0010 0.0004  0.4 mM M i s o n i d a z o l e : 0 LD/CV 1.01 206 0.74 LD 287 0.83 LD 413 0.58 LD 550 0.47 LD 688 L D / C V 0.31 1210 0.077 cv 1520 cv 0.019 1790 cv 0.0080 2110 cv 0.0015 2310 cv 0.00081  0.02 0.04 0.13 0.07 0.03 0.06 0.023 0.005 0.0036 0.0011 0.00024  5.0 mM M i s o n i d a z o l e : 0 LD/CV 0.96 161 0.80 LD 229 0.74 LD 322 0.69 LD 414 0.50 LD 517 L D / C V 0.36 852 cv 0.067 1050 0.025 CV 1210 0.0098 CV 1400 cv 0.0023 1530 cv 0.00098  0.04 0.04 0.10 0.06 0.03 0.04 0.012 0.006 0.0029 0.0010 0.00057  DOSE (Gy)  SURVIVAL  UNCERTAINTY  0.1 mM M i s o n i d a z o l e : 0 LD/CV 0.98 161 0.92 LD 236 LD 0.69 470 0.47 o 631 ° LD 0.48 814 L D / C V 0.36 1500 0.11 CV 1920 CV 0.032 2290 CV 0.0090 2710 cv 0.0020  0.02 0.25 0.04 0.04 0.06 0.06 0.06 0.011 0.0070 0.0006  1.5 mM M i s o n i d a z o l e : 0 LD/CV 1.00 138 0.87 LD 178 LD 0.78 252 LD 0.78 355 LD 0.57 458 LD 0.48 562 L D / C V 0.33 952 cv 0.088 1170 cv 0.040 1360 CV 0.017 1590 CV 0.0059 1740 0.00057 CV  0.00 0.08 0.03 0.03 0.03 0.03 0.06 0.012 0.015 0.008 0.0031 0.0003'  L  D  57  RESULTS AND DISCUSSION  4.1  UNCERTAINTIES. 4.1.1  error.  The  classes,  Experimental  error,  uncertainties  in  namely:  measurements, cell  1.  experimental partly  due  days to  metabolites,  etc.),  thus  not  experiments. error the  the  changes of  cells  included 3.  or  may  This  skill  of  error the  greater uncertainty the r e d u c t i o n of  is  not  cellular  be  of  the  in  each stage of  the  the  uncertainty  dose  This (pH,  cells cell  r e d u c e d by  improving  experimenter.  The  than those of  the  results  experiment  concentration  of  cycle.  error  for  errors  data.  This  the steps  between  skill  the  individual  of  experimental procedure set  This i s in part  through improved  and  Such  experiment  s e t B.  and is  experimental of  in  uncertainty  to repeat  variation  volumetric  (genetic)  estimates  the experimenter  three  points  e r r o r which leads to poor q u a l i t y  w i t h minimum o f  experimenter  to  into  and u n c e r t a i n t y  between  random.  experimenter  divided due  environment  nature  and  counting,  the  in  procedure  in cell  in  Experimenter  experimental  error  variation'  r e s u l t s from the i n a b i l i t y of  points. the  in  c a n be  experimental  fluctuations  may  variation,  experiments  'Biological  which  relative proportion are  2.  changes  these  Random  statistical  recognition.  biological  and  A  and have  due  to  experimental  procedure. Those  uncertainties  biological  variation  experimental variation  is  approximately  error dealt half  is  are  the  from  included  estimated  with of  resulting  after  for the  in  random the  experimental  uncertainty  error  and  estimates.  An  each s u r v i v a l measurement.  Biological  results  results  experiments  are  performed  obtained.  The  were d i s c a r d e d , m o s t l y  of due  58  to  bacterial  irradiation  contamination and  uncertainty  cells,  and  the  of  cell  assay.  to  for  from  The  dose  is  and  the  normally  estimate  Radiation  dose  radiation  delivered  by  cell  survival  low is  dose  survival,  about (63%  the  so  not  confidence  the  and  the  true  dose  t o be  rate.  less  the  curve  misonidazole  1% o f  ( F r i c k e and H a r t ,  is  the  in  the  it  is  The  standard  interval  in  the  cases.  uncertainty  steepest,  that  1966)  is,  c o n c e n t r a t i o n s where b e t a o f  delivered  An u n c e r t a i n t y  in  survival at  the  where  largest  equation  The  ferrous  t o be w i t h i n 5% o f  a c t u a l dose  the estimated dose.  largest  to  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 v a r i a t i o n  than  would produce survival  techniques  due  logarithm  d o s e r a t e d e l i v e r e d b y 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 s u l f a t e dosimetry  the  that  the  mean.  An  to  primarily  uncertainties  cell  mean  the  i s estimated in a l l  rate  plating.  of  dilution  the  after  during  parameters:  distributed  of  cells,  following  measured in  the  the  estimate of  a normal d i s t r i b u t i o n )  4.1.2  the  uncertainty  (Poisson)  the  of  as  of  incubation  each  survival  assume t h a t  survival,  deviation case of  cell  irradiation  during  misonidazole,  statistics  reasonable  or  estimated of  conventional counting  plating,  is  concentration  before  is  considered  i n t h e dose the  slope  doses  3 is large.  the  and  rate  of  the  highest  From  figure  -2 16  the  maximum  misonidazole 5.0  value  and  dose  survival, lower  rate and  At  will much  radiation  insignificant  beta  from t a b l e  mM m i s o n i d a z o l e .  the  of  less  doses.  and i s n o t  Misonidazole  equal  to  about  0.012  3 t h e maximum r a d i a t i o n  this  not  is  dose  it  can be  contribute at  lower  Uncertainty  more  than  the  dose  i n c l u d e d i n the e r r o r  solutions  were  shown t h a t  concentrations in  prepared  dose  Gray is  at 15.3  5.0 Gray  a 1% v a r i a t i o n  mM for in  10%  uncertainty  to  of  misonidazole  and  at  to  be  rate  is  considered  the  estimates. with  great  care.  The  59  concentration of misonidazole of  the  which  quoted  value  except  40 mM m i s o n i d a z o l e  have  dissolved  concentration repeated study.  in  in  for  was  these  than  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%  Uncertainty  in  few  experiments  prepared.  solutions,  quoted.  experiment  a  All  of  this  cell  Survival  survival  the  low  sources error'  dose of is  assay  error  B which provides  the  the  concentration  misonidazole  used  on  uncertainties.  on t h e will  in  be  the  contain  no  discussed  low  dose  assay.  cells.  for  for  Half  There The  to The  of  the  'possible error'  location errors  will  second s i g n i f i c a n t  cells  destined  to  vessel.  If  a cell  living  then  the  b e be source  survive  and  of  error cells  dying  cells  sd(death)  =  are  is  for  this  considered  deviation  are  that  'possible  for is  in  the  destined p,  of  'possible cannot  debris;  to  die  estimated  the  in  a  due  of  q,  of  binomial  Equation  N=number o f c e l l s . p=probability that a given c e l l i s a non-survivor. q=l-p=probability that a given c e l l is a survivor.  as  plating  and a p r o b a b i l i t y ,  distributed  are  estimates.  distribution  within  be  i.e.,  colonies  uncertainty  statistical  of  significant  either  sd(error),  the  dying  randomly  to  the  The e r r o r  two  error'  of  deviation,  (Npq)^  not  Thus any e x p e r i m e n t a l v a r i a t i o n  accounted  has a p r o b a b i l i t y ,  d i s t r i b u t i o n with standard  where  colonies.  data  look l i k e  counted as dead 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 , half  was  classification  colonies  the  not  misonidazole  survival.  colonies that of  in  estimates. standard  first:  c l a s s i f i c a t i o n day  viable  The  A  may  error  significant  a s s a y used t o measure  found at the recorded l o c a t i o n or they  lower  set  assay  depends  a  experimenter  of  set  misonidazole  in  be i n s i g n i f i c a n t and i s not i n c l u d e d i n t h e e r r o r 4.1.3  experiment  the  resulting  However,  in  4  60  From  equation 2 the r e l a t i v e  efficiency  d  (  P  LDA PS S  therefore,  s  d  _  )  and,  of the plating  sd(N) N  2  = (Npq) *  sd(N)  = E/2  (  p  s  )  L  = sd(error)  D  A  =  PS[(E/2N)  2  2  *  + (1/C-l/N)]*  Equation 5  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 .  5 was  used  to estimate  measured by t h e low dose There  sd(C) C  sd(C)= sd(death)  where,  assay.  i n the product  andt h e s u r v i v a l i s :  S  Equation  uncertainty  are three  the s t a n d a r d  deviation of survival  as  assay.  significant  sources  of error  i n the conventional  The e s t i m a t e o f t h e number o f c e l l s p l a t e d i s s u b j e c t t o a c o u n t i n g  u n c e r t a i n t y due t o t h e P o i s s o n d i s t r i b u t i o n o f c e l l s  i n t h e sample.  For a  P o i s s o n d i s t r i b u t i o n , t h e s t a n d a r d d e v i a t i o n can be e s t i m a t e d by,  sd(N)  Equation 6  = N=number o f c e l l s p l a t e d .  The  estimate  o f t h e number  of surviving  cells,  C , i s also  subject to a  c o u n t i n g u n c e r t a i n t y due t o t h e P o i s s o n d i s t r i b u t i o n o f s u r v i v i n g c e l l s i n the  sample.  A l s o , as s u r v i v a l  marginal  colonies  account  an e s t i m a t e  due  (colonies  decreases  with  c l o s e t o 50 c e l l s ) .  o f the standard  t o u n c e r t a i n t y i n the colony  t h e r e i s an i n c r e a s i n g number o f To take  this  into  d e v i a t i o n i n t h e number o f s u r v i v o r s  classification  was made f o r each  colony  '61  count  i n which t h i s  10% o f  the  u n c e r t a i n t y w a s deemed  survivors).  be e s t i m a t e d  Thus  =  where,  1,  the  and t h e  S  then,  d  (  for  and  S  e  of  Equation  sd(N) N  2  6 and 7 i t  can  the  7  to  product  of  the  plating  2  c a n b e shown  that:  Equation  2  these  experiments,  significant  is  at  least  e becomes l a r g e 8 simplifies  Equation  9 was u s e d t o  with  conventional in  in  due  _ = PS(1/N+l/C+e )^  sd(PS) _ _ C V L PS  deviation  colony count  2  uncertainty  sd(C) C  )  becomes  so t h a t e q u a t i o n  the  about  8  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 .  sd(PS)/PS  small S,  than  is:  CVL PS  P  from equations  the purpose  Therefore  2  survival  where,  N=C/PS,  the  (C + C e )  relative  sd(PS1  For  of  e=estimated r e l a t i v e e r r o r survivor miscount.  equation  efficiency  standard deviation  (greater  by,  sd(C)  From  the  significant  the  C is  at  0.10  less  than  survival for  large  or  levels S  about of  150,  less  (approaching  10%. and  and 1/N becomes i n s i g n i f i c a n t compared t o  1/C,  to:  Equation  2  estimated the  normalized  than unity),  I = MAX[0.10, ( l / C + e ) ]  assay.  P=0.6,  Note  uncertainties  that  survival,  in it  order is  also  of to  survival estimate  necessary  9  as  measured  the  standard  to  take  into  62  account the u n c e r t a i n t y which i s obtained The assays: account,  the  the  is  a  (the  in  uncertainty S=l),  low dose  in  and i f  assay,  9 when t h e  comparative  uncertainty  r a d i a t i o n dose, in  from equation  following If  i n the n o r m a l i z a t i o n  E,  the P  LDA S  _  s  d  L D A S  ~  where, since,  Similarily Q,  is  if  taken  miscount,  e,  JL (  N  into  account  i n the  survival,  then  relative  the  taken 5  for  d  (  S  )  C LVV LL  1 _ N LDA  S  91 Equation  equation  9),  assay i s kept dish  is  uncertainty  if  the  small,  error  in  the  10  (approximately) l e v e l f o r the low  due  and i f  (approximately)  where,  2  efficiency, to  the  survivor number  independent  conventional  assay  1  = (jL-r± + s + 11 ) * o  in  1 _ 1 ] * N P ~ N LDA LDA  i n the conventional assay p l a t i n g  petris  1  zero  uncertainty  approximately:  S  into  » *  LDA  conventional per  8 is  sd(P) P  2  r^Ltl. + n  (using  colonies  two  classifications  relative  L D A  the u n c e r t a i n t y  these  is  equation  'possible error' the  of  P,  C=NPS N =number of c e l l s p l a t e d , i s independent of the s u r v i v a l low dose a s s a y .  surviving  equation  from  then  efficiency),  5 is:  [__1 N PS LDA  _  analysis  efficiency,  estimated  small,  sd(PS) PS  =  therefore,  plating  (plating  S=l.  uncertainty  is  kept  the low dose assay from e q u a t i o n  S d ( S )  survival  t h e number o f  is  factor P  +  B  +  ^-J  Equation  Q=conventional assay 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 .CVL^. _ , ^ ^ i r r a d i a t i o n dose i f C i s independent of s u r v i v a l .  11  of of  from  63  The  two  survival  assays  can be  compared  by  estimating  number o f c e l l s w h i c h m u s t b e p l a t e d i n o r d e r This  is  done  equation  Now, a  by  survival  N  CVL _  N  LDA  =  [  of  from  1  +  S  )  /  S  with  the  Q,  assay  the  assay,  error'  i s estimated  for  the conventional  due  to  Even  t h r o u g h more  is  collected.  or  the  assay  total  'experimental  error'  and  straightforward  way t o  several  times.  combination  if  survival Most  in  5 for  at  each  of  the  from  12  dose  survival  o f t w o o r more d a t a p o i n t s .  the  of  are  as  that  fifteen  assay  plating  estimating  the  t e n t i m e s a s many  as a c c u r a t e a  point.  assay  result  data  after  survival  equation  a l l of is  in  due  experiment  these data points  9  error  the  data  to  the  The  survival  misonidazole  by  'experimental or  variation'.  of  measured  The a d d i t i o n a l  uncertainty  each  is  The r a n d o m  low dose  in  If  a result  Survival  be e s t i m a t e d  for  cells  at  S=0.8.  assay.  total  many  approximately  approximately  'biological the  0.90 so t h a t  as  a c c u r a t e means  uncertainty the  S=0.9  survival.  can only  estimate  sd(S)  the conventional  f o r each s u r v i v a l  The  the  for  assay to y i e l d  conventional  from equation  times  as a c c u r a t e  l e v e l of  uncertainty  'biological variation'  measure  seven  and  by t h e c o n v e n t i o n a l  low dose  with  0.60 and P i s about  i n s u s p e n s i o n s o t h a t Q=P,  either  10  C VL  to yield  are required.  total  the  2 S ]  as t h e low dose assay a t a s u r v i v a l The  of  Equation  approximately  was i m p r o v e d  are required  4.1.4  equation  result:  Q i s about  low dose  ratio  t o a c h i e v e t h e same a c c u r a c y .  + 1]  "  P  the conventional  density of c e l l s cells  (  +  S=0.5  a s many c e l l s  efficiency,  [2/Q  experiments,  level  by  attainable times  sd(S)  LDA 11 w h i c h g i v e s t h e f o l l o w i n g  f o r these  required  equating  the  most is  to  concentration set  B  is  a  are  normally  64  distributed  about  t h e mean  be e s t i m a t e d as f o l l o w s  sd(S.)  =  1  survival  the  (Bevington,  _ ' (q  _q  standard  deviation  If  each  data  } 2  *  v  has  the b i o l o g i c a l v a r i a t i o n  can be n e g l e c t e d and t h e follows  (Bevington,  l/sd(S ) ±  where,  is  Equation  an  associated  s m a l l compared t o  standard deviation  the  equation  standard 14  experiment the  the  value  the experiment  =  [l/sd(S )) k  greater  the  significant  days,  the  experiment survival  deviation  in  uncertainty  in  of  sd(S^)  set B data,  was  than  that  error  it  can be e s t i m a t e d  as  Equation of kth data  cannot  survival  from  for  equations  K.  ).  survival  be  less  each of  13  between  from  This  survival This  i s repeated at l e a s t three estimated  14.  than  X-ray  dose  survival and  14.  is In  measurements  point  estimated general,  made  also points  13.  from  from equation  Therefore  is  on  out  times, the uncertainty equation  sd(S)  was e x p e c t e d a n d  result  14  point.  the t o t a l estimated u n c e r t a i n t y  from equation  variation  best  experimental  2  survival  'biological variation'.  is  sd(S  1969):  s e t B w i t h t w o o r more m e a s u r e m e n t s  maximum  error,  in survival  sd(S^)=experimental error  the  13  dose  experimental  sd(S.)=uncertainty i n averaged a t i t h dose p o i n t .  Since  can  i  k=l,N k I N(N-l)  point  survival  1969):  S^=Survival, kth data point. S.=Average s u r v i v a l at a g i v e n X - r a y misonidazole concentration. N=number o f d a t a p o i n t s .  However,  in  due  of as for 13 to  different that  i n the the  if  an  average tedious  65  uncertainty  calculations  used  to estimate  experimental  error  need  n o t be  carried out. If  o n l y one d a t a p o i n t  i s obtained  misonidazole  concentration  uncertainty  (which  variation') suitable  another  includes  i s required.  f o r a p a r t i c u l a r r a d i a t i o n dose and means  'experimental  To d o t h i s  f i t to the survival  of  estimating error'  and  i t was assumed t h a t  data.  The most  the  'biological  equation  l i k e l y values  total  3 was a  f o r alpha and  2  beta  were  estimated  Bevington,  by m i n i m i z i n g  the variable  X  of  equation  15  (after  1969).  S -S(a,b) sd(S )  2  '  1  and  S  i ^ ^ sd(S.) l s  1  1  i  2  E q u a t i o n 15  J  = the residuals.  a , b  r-i v-v , - a D - b D S(a,b)=10  where,  A  E q u a t i o n 16  2  S.=survival, i t h data point. sol(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=radiation dose.  The  residuals  and p l o t t e d in  (equation  against  the residuals  spread  in  survival.  the data  that  standard  points  gave  s e t A gave  a mean  standard  of  calculated  Neither  times  of  a mean  0.5±2.5.  deviation  the  that  normally  residuals o f 0.1±2.3  Thus  i t  o f each data p o i n t  is  on s u r v i v a l ;  the total  the experimental  were  f o r each  survival,  t h e mean n o r t h e s t a n d a r d  indicated  the residuals  deviations  experiment  then  showed a s i g n i f i c a n t dependence  s u r v i v a l was two t o t h r e e assumed  16) w e r e  were  uncertainty  reasonable  t o be about  i n the  I t was t h e n  a n d t h e means a n d  calculated.  and t h e data  deviation  however, t h e  uncertainty.  distributed  S^,  The  data  o f experiment  to estimate 2.5 times  the  of  set B total  the estimated  66  experimental uncertainty  sd(S^)  For standard  = 2.5 x  experiment  variation  of  since had  experiment  the  experimental  be  equation data  error.  In  deviation  these  cases For  used data  estimate of  standard  point,  o t h e r w i s e t h e maximum v a l u e o f  to  For was  sets  with  data  sets  equations  if  total  than  with  only  the  one  one  estimated  variation'  set B equation  deviation  the  'biological'  as  'biological  17  more  or  estimated  the  Eqn.  estimate  experiments,  experiment  as t h e  4.2  total  for  account.  insignificant. the  was  between  into  standard  17  point  variation taken  total  c o n s i d e r e d t o be  A  each  the  to  follows:  (estimated experimental uncertainty)  set  deviation  experiment  as  17 was  t h e r e was o n l y  one  was used data  13 a n d 14 was u s e d .  RESULTS. 4.2.1  Method o f parameter  set B are  tabulated  radiation  dose  and  survival.  The  most  estimated  by  the  standard  total  radiation  the  analysis. standard  table  likely  minimizing  it  the  deviation  was  estimates If  the  deviation  2.  values  the  sd(S^)  to  and  then  the  average  of  equation  X  equation  15  where  of  2  beta  times This  but  S^,  Since the for will  greatly  are  variable  not  X  2  has  each  at  were is zero  misonidazole significantly  simplifies  normally  3  sd(S^)  survival each  at  measured  beta  unity.  the  survival  and  several  values,  is  experiment  alpha  survival.  measured  alpha  survival  of  variable of  set B the  concentration  constrained of  The s u r v i v a l d a t a o f  For experiment  misonidazole  d o s e was g e n e r a l l y  concentration affect  in  estimation.  the  data  distributed  with  a  chi-squared  67  distribution  f o r w h i c h t h e number o f d e g r e e s  n o n - z e r o dose p o i n t s estimated alpha  i n t h e d a t a s e t minus t h r e e ,  f o r each d a t a s e t ) ,  and b e t a  (Bevington,  o f freedom  are  and t h e most  those  which  in  p r o g r a m was w r i t t e n  s u r v i v a l data on an a r r a y NxM, a n d t h e r a n g e divide  increments, and  o f the parameters value  of  X . 2  was e s t i m a t e d a s f o l l o w s :  at  2  of X  was t o f i r s t values  2  the values  o f a l p h a and b e t a v a l u e s .  range  t h e range X  to calculate  o f a l p h a a n d b e t a was d e f i n e d  data using  the array  alpha  the  calculate  survival  were  1976). 2  would  values  t h e minimum  The minimum v a l u e o f t h e f u n c t i o n X ( a , b ) computer  (since two parameters  likely  result  i s t h e number o f  of  of beta each  alpha into  alpha  equation  into  N  M values and b e t a  15.  of X  separated  by t h e user.  The p r o g r a m  values  separated  separated  by equal  value  in  0 . 0 0 5 Gy ^  2  a n d 61 b e t a  by  equal  increments  the array  A second program was w r i t t e n  l o c a t e t h e minimum o f X  by  f o r a set of  2  The s i z e o f t h e a r r a y ,  v a l u e s on a graph o f alpha versus b e t a .  approximately  A  from t h e to  contour  The p r o c e d u r e  using  an array  values  used o f 41  separated  by  -2 0 . 0 0 0 5 Gy  .  This  and b e t a p a r a m e t e r s intervals alpha  generally  allowed  i n order  t o contour  on t h e e s t i m a t e s  values  -2 0 . 0 0 0 1 Gy -1 ±0.001 Gy  separated was u s e d i n alpha  sufficient  0 . 0 0 1 Gy ^  range  Next  a n d 31 b e t a  located  t h e minimum X -2 a n d ±0.0001 Gy i n beta.  2  a finer values  with  grid.  parameters  w a s much  larger  than  the uncertainty  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  programs  and $150.00 t o c a l c u l a t e  calculating the array  of X  2  the results).  values.  Cost  a  Greater  considered unecessary because t h e observed u n c e r t a i n t y these  in  the  alpha  t h e 70%, 9 5 % , a n d 9 9 . 9 % c o n f i d e n c e  o f these parameters.  by to  for  array  o f 21  separated  by  resolution  of  r e s o l u t i o n was  i n the estimates of introduced  by t h e  (about $50.00 t o d e v e l o p t h e The major  c o s t was due t o  c o u l d be reduced by u s i n g  a more  68  efficient routine  function written  difficulty  minimization  f o r the University  l a y i n the large  alpha and b e t a near its  major  axis  where  X  I t i s very  i s not large  2  result  of the parameters  If X  X  respect  one such  IBM 370 c o m p u t e r .  would  2  tried  take 2  difficult  i s an e l l i p s e  t o the alpha  to constrain alpha region  minimization being  Consequently  L  Q  (equation  G  suitable  18)  would  i n t h e exponent  of the survival  f o r function minimization  x  2  error  "  i = 1  '  The p r o b l e m w i t h e q u a t i o n survival  the important  not  a chi-squared  the  quality  been  of  equation.  of  high  distribution. (P(X ,v)) 2  However,  estimating  2  be  E q u a t i o n 18  This  survival equation  and confidence  t h i s may h a v e f i t  i n the logarithm of  is particularily  data.  Consequently  18 w a s u s e d intervals  of  alpha  X  2  L  warrant  data  such an a n a l y s i s .  analysis  (e.g.,  Chadwick  Many  researchers  and Leenhouts,  results  would  less  and b e t a  use equation 1981;  0  is  G  the v a l i d i t y  led to a quicker,  values  noticeable  Kellerer  of  have  expensive 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 h a v e b e e n s u f f i c i e n t l y to  more  2  the uncertainties  If  the best  function  and would  ]  distributed.  low dose,  f i t  jeopardized.  method  18 i s t h a t  are not normally  for  slhsT]  N [  rapidly  was a  algorithms.  log(S.)-aD-bD  =  L 0 G  so  would  t h i s more a n a l y u t i c a l  The u n d e r f l o w  increase  region  The p r o g r a m  was abandoned.  not  axes  axis of  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 used i n s t e a d , t h e c h i - s q u a r e d 2  of with  and b e t a  and b e t a t o a  is elliptical.  errors.  The  on i n r e g i o n s  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 minor  then terminate due t o underflow of function  Victoria  which  with  since this  method  unsuccessfully  Since the function X  diagonal  occur very  2  I  of  values  t h e minimum.  running  large values of X the e l l i p s e .  routine.  18 f o r  normal their  and B r e n o t ,  69  1974) . high  However,  survival The  and  squares  those  some  and  which  has  not  been  justified  T h i s has been l e f t  squares  method  deviation  use  levels.  least  approach  its  method  may  of  wonder  assumes  for  this  that  each  therefore  defines  the  minimize  the  function  X  fit  is  not  has  values of  2  of  data  with  consideration.  was  point  best  case  estimation  method  data  the  future  parameter  why  in  a  much  used.  the of  The  same  alpha  equation  easier least  standard  and b e t a  as  with  the  18  LOG  uncertainty  term set to  estimate  the  of  a constant.  standard  deviation  In p r a c t i c e t h i s approach favours more  accurate  squares as  fit  low dose  failed  at  by zero  miserably  parameters, magnitude  the  in  Otherwise  its  than  the  parameters  probability t a b l e C-4 of 4.2.2  of  an  the  normally  alpha  of  of  be  unity. the  set  is  The  which  were  at  variation  in  allows  for  if  the  If  a good a p p r o x i m a t i o n )  for  experiment  and  if  fit  in of  of  an  is  a  the  order  of  parameters. estimation  the X ,  survival the  2  minimum v a l u e ,  function  a r e shown i n t a b l e  squares  parameter  The minimum v a l u e s this  the  these  variation  least  if  deviation least  the  parameters  only  least  an  1976).  B the  the  but  standard  which y i e l d  value  method,  estimation.  this  (Bevington,  experiment  reserved  which  confidence  (or  conditions  1976)  to  of  observed  should  this  of  minimization  limits  and b e t a  function  70% c o n f i d e n c e l i m i t s o f  is preferable.  Bevington, Method  the  this  parameters.  data  estimates  approach  obtaining  m a t h e m a t i c a l model under  the  constrained  approach  u n c e r t a i n t y w i t h dose of  For  uncertainty  smaller this  was  the  of  the lower q u a l i t y h i g h dose d a t a over  chi-squared  dose  giving  Consequently only.  data.  of  gave r e s u l t s w i t h i n t h e  estimated  survival  Then t h e v a l u e  good  values and  fit  the (from  3. equation the the  d i s t r i b u t e d with t o t a l standard deviation  3 is  survival survival  sd(Sj  the of  CHO  data,  then X  2  correct  S^, of  cells are  70  TABLE I I I  Table I I I :  CHI-SQUARE F I T RESULTS.  The v a l u e o f t h e c h i - s q u a r e d f u n c t i o n , e q u a t i o n 15, was c a l c u l a t e d f o r a g r i d o f a l p h a and b e t a v a l u e s f o r t h e s u r v i v a l d a t a o b t a i n e d f o r each concentration of misonidazole. The f u n c t i o n was contoured a t the i n t e r v a l s shown i n t h e t a b l e , c o r r e s p o n d i n g t o c o n f i d e n c e i n t e r v a l s i n t h e e s t i m a t e s o f the a l p h a and b e t a parameters o f 70%, 95% and 99.9%. These c o n t o u r s a r e shown i n f i g u r e s 14 through 17. The v a l u e s o f a l p h a and b e t a which gave a minimum v a l u e o f t h e c h i - s q u a r e d f u n c t i o n a r e shown a l o n g w i t h t h e minimum value. The p r o b a b i l i t y o f o b t a i n i n g a v a l u e o f c h i - s q u a r e d g r e a t e r than o r e q u a l t o t h i s v a l u e ( i f e q u a t i o n 3 i s the c o r r e c t f i t t o t h e data) was determined from t a b l e C-4 o f Bevington (1976). T h i s p r o b a b i l i t y (PBY) i s shown i n t h e l a s t column o f t h e t a b l e .  MISONIDAZOLE CONCENTRATION (mM)  X  CONTOUR VALUES  ALPHA (/Gy) +0.001  BETA (/Gy ) ±0.0001  MINIMUM VALUE OF X  PBY  EXPERIMENT SET A RESULTS: 0.0 28.3 37.8 52.5 0.05 9.52 15.5 26. 2 0.10 19.5 27.5 40.8 0.25 18.4 26.2 39.2 0.50 19.5 27.5 40.8 1.0 25.9 35.2 49.8 3.0 28.3 37.8 52.5 5.0 27.2 36.5 51.2 10.0 9.52 15.5 26.2 40.0 9.52 15.5 26.2  0.020 0.026 0.053 0.035 0.052 0.018 0.022 0.059 0.082 0.055  0.0026 0.0022 0.0022 0.0030 0.0059 0.0072 0.0170 0.0092 0.0129 0.0074  25.8 8.5 5.5 3.0 16.3 35.8 25.9 12.8 3.75 9.37  0.31 0.21 0.99 0.99 0.36 0.02 0.14 0.93 0.71 0.16  EXPERIMENT SET B RESULTS: 0.0 11.8 18.3 29.6 0.1 10.7 16.9 27.9 0.4 10.7 16.9 27.9 1.5 10.7 16.9 27.9 5.0 11.8 18.3 29.6  0.029 0.052 0.042 0.039 0.027  0.0019 0.0018 0.0043 0.0078 0.0118  0.55 1.39 0.79 0.73 0.62  0.82 0.21 0.60 0.65 0.76  2  2  71  equation  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 a n d t h e p r o b a b i l i t y  a s e t o f data such t h a t determined. the  (Bevington,  number  alpha  X  of  1976).  non-zero  and b e t a  exceeds a s p e c i f i e d value  2  T h e number  radiation  dose  are not allowed t o vary  used  t o determine  interval. of  F o r example,  freedom),  than  the values  X  18.3 with  probability  than  probability  0.01 giving  respectively.  0.05,  the  and contoured  was  a  set of  misonidazole 14  limits)  limits),  17  f o r experiment  range  in  resolution  and  X  4.2.3 of  the data  experiment estimated  sets sets  closed  only)  the parameters  Results  s e t being curves  2  than  is  greater  29.6  with  intervals  limits.  on t h e l o c a t i o n o f t h e contoured.)  (error  f o r experiment  The v a l u e alpha  The r e s u l t  ellipses)  for  s e t A and f i g u r e s  limits), of X  and beta  f o r each misonidazole  2  a n d 18  (99.9%  16 (70%  drawn and  i n table  concentration  figure  confidence  f o r the contours  a r e shown  each  3.  The  i s ±0.005 Gy ^  i n beta.  o f parameter  obtained  experimental  greater  depended  and confidence  f o r 0 mM a n d 5 mM m i s o n i d a z o l e were  0.3, X  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  (95% c o n f i d e n c e  -2 i n a l p h a a n d ±0.0005 Gy  is  2  (ten degrees  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  s e t B.  of the grid  (1976) w a s  f o r a 70%, 95%, a n d 9 9 . 9 % c o n f i d e n c e  f o r the data  concentric  limits  do n o t  a t t h e 70%, 9 5 % , a n d 9 9 . 9 % c o n f i d e n c e  concentration.  (70% c o n f i d e n c e  confidence  the  function  three  and t h e r e f o r e  Table C-4 of Bevington  (The r a n g e o f t h e a l p h a a n d b e t a p a r a m e t e r s 2  i s now  The p a r a m e t e r s  70%, 9 5 % , a n d 9 9 . 9 % c o n f i d e n c e  2  minimum o f t h e X  freedom  one.  11.8 with probability  The f u n c t i o n X ( a , b )  61 b e t a v a l u e s  2  of  f o r a data s e t with eleven data points  i s greater  2  of X  minus  but are fixed  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 .  obtaining  can be q u a n t i t a t i v e l y  o f degrees  points  of  from equation  standard  deviation.  estimates.  The r e s i d u a l s  concentrations  16 w i t h  sd(Sj  Qualitatively  from  equal the  both  to the  residuals  72  tended  to  0.1±2.3  normally  standard  deviation was  be  deviation  f o r experiment  on t h e average  uncertainty. values  for  s e t B.  i s a slight  than  those  i n the region  of  T h e mean  experiment Thus  approximately  There  greater  S(a,b))  distributed.  the t o t a l  tendency  predicted  greater  than  3  less  0.05.  can be a t l e a s t p a r t i a l l y  difference  between  conventional set  that  measured  measured  therefore equation less  the  which  w a s 0.12±0.08  standard  the  to give  values  3 and t h e c o n v e n t i o n a l value.  s u f f i c i e n t l y small that source  of the discrepency  Qualitatively  marginally  viable  located  on t h e l o c a t i o n  locate  than  the  than of  at  lower  day, (poorly  for  and t h e  experiment  s e t B.  marginally low dose  tend  t o give  Thus  healthy,  appropriate  survivals,  proliferating  the  higher  than  assay  survival  in survival  Quantitative  growing  those  survival  would  some  study could corrections  cells  cells)  of  which  a r e more resulting  values  measurements reveal f o r the  t h e d i s c r e p e n c y may b e e x p l a i n e d b y t h e l a r g e r cells  than  than t h e v a l u e p r e d i c t e d by  discrepency  and p r o v i d e  greater  assay  deviation  The  greater  i t was i g n o r e d .  (S^  survival  explained by t h e observed  f o r experiment  assay would This  t o have  by t h e l o w dose  assay.  experimental  i n the region  a s s a y was a g a i n  conventional  3  standard  uncertainty  0 . 2 and l e s s  standard  deviation  survival  the predicted  S(a,b))  measured  by t h e l o w dose  by  tend  than  data. of  assay  A a n d 0.18±.09  survival  is  the survival  experimental  equation  by equation This  a n d 0.5±2.5  f o r data points  by  than  t h e d i s t r i b u t i o n was  the estimated  predicted than  (S^ l e s s  set A  2.5 times  survival  of  number  were  difficult in  a  not to  higher  73  estimate of  s u r v i v a l w i t h the  P(X ,v)  is  z  (equation and  15)  the  if  degrees  of  probability  the  standard  survival  deviation  freedom.  The  s e t s A and B a r e g i v e n which  fall  since  the  values  of  (equation  16)  best  fit  linear-quadratic  Note or  of  the  that  less  three  than  outside  of  values of  but  the  0.02.  There  the  in a rather  confidence  on  and  once.  measured  'biological  variation'  uncertainty  was  (variation  within  is  only  The  0.05,  for  and P ( X , v ) = 0 . 2 2  is  mean  of  50%.  (30%)  This  are  10.0,  of  for  of  a  these  given very  low  standard  sum o f  the  average. than  set  factors.  Thus  only  rough  falling  as  this  estimates.  a n d 4 0 . 0 mM m i s o n i d a z o l e in  these  was  experiments)  estimated  where  cases  misonidazole,  not  not  assumed,  and that  subject  therefore  the the  to the  uncertainty is  P(X ,v)=0.7  40 mM m i s o n i d a z o l e .  is  concentrations  and  variation  high  assumption  Note  'experimental'  biological  0.98  and v e r y  deviation  so the  the  data  be  about  greater  2  to  chosen  the t o t a l standard d e v i a t i o n  between  5 mM  P(X ,v)  2  is  was  on  of  experiment  the  value  mean  P(X ,v)  the t o t a l standard d e v i a t i o n ,  survival  the  forcing  2  experiment  distributed  1),  have  4% c h a n c e  unrelated  as  normally  2  number  of  data points  X =x  with  the  results  a reasonable  occurence  experiment  2  be  experimental  The  estimated an  of  set A g i v e s a set of  (equation  sets  (variation  P(X ,v)=0.2  misonidazole,  would  data  2  were  v  the  individual  yield  P(X ,v)  the  control).  the  curve  other  s u r v i v a l curves for only  Experiment  crude estimate of  contours  for  2  value  distributed  parameter  expected  to  2  estimated  depends  minimum  normally  P(X ,v)  i s an i n d i c a t i o n t h a t  2  to  X  ten  bounds.  P(X ,v)  rather  results  of  these  proportional  dose  residuals,  a  s e t s w h i c h c o n t a i n e d more t h a n o n e  deviation  residuals  are The  3.  standard  the  obtaining  of  the  data  that  squares  of  sd(S^).  about  case of  assay.  values  in table  reasonably  expected i n the  low dose  2  These a r e  easier for  to  10 mM  reasonable  74  values  and t h e r e f o r e  Experiment  set B gives  with the hypothesis The  reasonable  data  sets)  properly  equation  indicates  estimated.  2  Figure  obtained that  of  was made.  and b e t a of  alpha  rough  based  estimate  confidence  limits.  evidence of  this.  their  3)  axes  The  the  the  the  15,  16,  is  estimate  the of  and  area of  residuals  the  from  show  respectively for  observed standard the  between  deviation  variation  r a d i a t i o n dose and m i s o n i d a z o l e  the  in  points  was  the  i n the  The  with  of  contours  respect  to the  directly  on  w h i c h was o n l y  the  experiments  the  0.25  mM,  a of  the  70%  contours  shows a s i g n i f i c a n t than  the  between  depends  size of  greater  total  intervals  contours  s e t A.  of  making  A.  correlation  all  is  increase wherease  alpha. estimated  the  of  data  survival  concentration).  70%,  s e t B.  estimates in  set  ellipse  experiment  the  five  are a rough estimate of  variation  data.  the  contour  the data p o i n t s ,  misonidazole  17  for data  diagonally  strong  The c o n f i d e n c e c o n t o u r p l o t of  to^the  s e t A r e s u l t s , by  experiment  Thus t h e c o n t o u r s  concentrations  correlation  measurements  in  estimated  oriented  demonstrating  The d e g r e e o f  confidence contours  of  the  for  shows no s i g n i f i c a n t i n c r e a s e i n  Figures  0.8  consistent  a much b e t t e r e s t i m a t e o f  estimated  (equation  on  s e t s A and B.  the p l o t  to  deviation  standard deviation of  experiment  for  (0.2  2  standard  the  and b e t a .  the  beta  P(X ,v)  Consequently  of  with  axes  the estimate of  in  for  70% c o n f i d e n c e  a plot  elliptical  estimates  3 i s a s u f f i c i e n t l y good f i t  14  is  data.  which are  experiment  tend  alpha  3)  a r e much more r e l i a b l e t h a n f o r  a l p h a and b e t a  the  to t h i s  (table  2  each data p o i n t  parameters t o be  the  P(X ,v)  fit  In comparison w i t h experiment  s e v e r a l measurements  and P ( X , v )  a set of  that equation  values  standard deviation  3 i s a reasonable  and  99.9%  Once a g a i n a  strong  alpha  points  at  95%,  each  and  beta.  The  was  obtained  from  data  point  (each  This estimate  will  75  T  I  0.00  1  I  I  1  !  0.05 alpha  F i g u r e 14:  0.10  (/Gy)  ALPHA AND BETA CONFIDENCE CONTOURS, EXPERIMENT SET A.  The 70% c o n f i d e n c e l i m i t s o f t h e e q u a t i o n 3 parameters, a l p h a and b e t a , were e s t i m a t e d u s i n g t h e c h i - s q u a r e d t e s t . A different confidence interval was estimated from the data f o r each 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 as i n d i c a t e d on t h e graph. Those d a t a s e t w i t h a minimum v a l u e o f c h i - s q u a r e d g r e a t e r than t h a t which would y i e l d a 70% c o n f i d e n c e i n t e r v a l a r e i n d i c a t e d w i t h a c r o s s (X).  76  ' Gy  CM  ca CD jQ  0 . 20  0 . 1 0  0 . 00 0.00  0.0 5 alpha  F i g u r e 15:  .0.10  ( / G y)  ALPHA AND BETA 70% CONFIDENCE CONTOURS, EXPERIMENT SET B.  The 70% c o n f i d e n c e l i m i t s o f t h e e q u a t i o n 3 parameters, a l p h a and b e t a , were e s t i m a t e d u s i n g the c h i - s q u a r e d t e s t . A different confidence i n t e r v a l was e s t i m a t e d from the d a t a f o r each 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 as i n d i c a t e d on the graph. These c o n f i d e n c e i n t e r v a l s a r e much s m a l l e r than those e s t i m a t e d f o r experiment s e t A, f i g u r e 14. Thus t h e r e s u l t s f o r experiment s e t B a r e much more a c c u r a t e .  77  CM  (3 0 . 2 Oh  z  °-  10  O.OOh  0.0 0  0 . 0 5 . 0 . 1 0  alpha  (/.Gy)  Figure 16: ALPHA AND BETA 95% CONFIDENCE CONTOURS EXPERIMENT SET B. The 95% confidence l i m i t s of the equation 3 parameters, alpha and beta, were estimated using the chi-squared test. A different 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.  78  CM >»  CD  0 . 20 -  CO  JQ  0.00  — »  0.0 0  0.05  alpha  F i g u r e 17:  .0.10  ( /G y )  ALPHA AND BETA 99.9% CONFIDENCE CONTOURS EXPERIMENT SET B.  The 9 9 . 9 % c o n f i d e n c e l i m i t s o f t h e e q u a t i o n 3 parameters, a l p h a and b e t a , were e s t i m a t e d u s i n g the c h i - s q u a r e d t e s t . A different c o n f i d e n c e i n t e r v a l i s e s t i m a t e d from the d a t a f o r each 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 as i n d i c a t e d on the graph.  79  approach  the  true  standard  each data p o i n t . the in  true  confidence  beta  plot  for  experiment  A  when many m e a s u r e m e n t s  are  contours  estimates  confidence  limits.  increase in  (figure  are  The c o n t o u r p l o t  concentrations  significant  set  14)  are  shown i n  of  the  figure  most  18  alpha.  results  are  values  (experiment  70% c o n f i d e n c e  beta i n various  likely  s e t A)  interval.  ranges  shown  in  of  of  than  0.1  misonidazole  table  IV.  mM,  The r e s u l t s less  alpha  and  figures  from  averages  c o n c e n t r a t i o n s were  The  two  sets  of  results  are  of  for  greater  experiment a c c u r a c y due  discussed in section 3.3.3. does  not  change  uncertainty.  s e t B were o b t a i n e d to  improvements  results  clearly  but  remains  increases  in  c o n c e n t r a t i o n b e t w e e n 0 . 1 mM a n d 5 . 0 mM. i n c r e a s e was n o t points  and  more  from fewer the  The r e s u l t s o b t a i n e d  significantly  Beta  in  of  misonidazole  3  s e t B) of  within  are with  alpha  and  are  The  clearly  experiments  of  and  method  as  alpha  experimental misonidazole  o r upper bound o f  a c c u r a t e l y measured by t h e s e e x p e r i m e n t s . concentrations  of  uncertainties.  within  interval  The n a t u r e  sets  a l p h a show t h a t  constant the  earlier  table  experimental  for  the  estimated.  c o n s i s t e n t w i t h each other w i t h i n the estimated experimental The  whereas  Both  (experiment  the  of  uncertainties.  beta  19  for  increase  from the  accurate.  and f i g u r e  From t h e s e  made  shows a s i g n i f i c a n t  greater  clearly  reasonable  are c o n s i s t e n t w i t h i n the estimated experimental  Plots  the  the  misonidazole  shows no  results  Thus  deviation  More h i g h this  range  this dose are  required.  4.3  SIGNIFICANCE Two  sets  of  OF THE R E S U L T S . experiments,  measure t h e s u r v i v a l o f  A  CHO c e l l s  and  B,  were  performed  consecutively  s u b j e c t e d to v a r i o u s doses of  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 .  Experiment  set A provided  to  X-radiation preliminary  80  Figure 1 8 :  ALPHA AND BETA DEPENDENCE ON MISONIDAZOLE CONCENTRATION, EXPERIMENT SET A.  Survival curves were obtained at various concentrations of the r a d i o s e n s i t i z e r misonidazole. The parameters alpha and beta of the linear-quadratic equation 3 were estimated f o r each curve. The estimated 70% confidence l i m i t s from figure 14 are shown.  81  Figure  19:  ALPHA AND BETA DEPENDENCE ON MISONIDAZOLE CONCENTRATION, EXPERIMENT S E T B.  Alpha and b e t a were estimated as i n figure 18. T h e 70% confidence limits from figure 16 a r e shown. These data a r e c l e a r l y much more r e l i a b l e than the experiment s e t A data of figure 18. Beta clearly increases with misonidazole concentration, alpha remains c o n s t a n t within the experimental uncertainty.  82  TABLE  Table  The  IV:  THE RANGE OF THE PARAMETERS ALPHA AND B E T A .  range  of  minimization,  parameter alpha  IV  the  parameters  experiment  s e t s A and  [MISONIDAZOLE] a l l concentrations  beta  0-0.1  beta  5.0-40  mM mM  alpha  and  beta  as  estimated  by  chi-squared  B.  Experiment 0.04210.010  set A Gy"  Experiment 0.03910.010  1  0.002310.0003  Gy"  0.006510.0056  Gy  2  -2  set B Gy"  1  0.001910.0002  Gy"  0.011810.0024  Gy  2  -2  83  data The  which  has n o t been  survival  survival over  data  values  experiments  q u a d r a t i c model  (equation  estimate the parameters  'best'  experiment  and m i s o n i d a z o l e performed  was n o t  for  averaged.  i n table  II.  c o n c e n t r a t i o n were  experiment  s e t B.  i n figures of  show  Results are tabulated  The  averaged A  3) w a s 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  estimates  s e t B data  The s u r v i v a l  set B i s tabulated  a l p h a and b e t a .  IV and graphed  The  here.  f o r experiment  f o r each dose  the three  and t a b l e  tabulated  linear sets to  i n table  III  14 t o 1 9 .  the  survival  a significant  curve  trend  parameters  for the  i n the high-dose  survival -2  parameter,  beta,  which  increase  from  0.0019±0.0002 Gy  to  -2 0.012±0.002 Gy Uncertainties  misonidazole  are estimates  confidence. parameter,  between  There alpha,  is  no  which  The s u r v i v a l  against  i n figure  between  the  data  t h e maximum  significant  remains  confidence. dose  of  concentrations  data  20.  trend  constant with  at  these  range in  of  0 . 1 a n d 5 . 0 mM.  of  beta  the  within  l o w dose  70%  survival  0.039±0.010 Gy ^ w i t h i n 70%  best  f i t curves  are  plotted  A t low doses 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  points  obtained  with  different  concentrations  of  misonidazole. 4.3.1 estimates  MISONIDAZOLE DMF d e p e n d e n c e of  alpha  concentrations to  0.8.  survival  and b e t a  of misonidazole  In table V these curves  from  of  Chinese  experiment  were  results  on s u r v i v a l  level.  From t h e ' b e s t '  s e t B DMF's  of  calculated at survival  a r e compared t o v a l u e s  hamster  cells  treated  with  the various  levels  obtained  of 0.01 from t h e  misonidazole  and  X - r a y s b y Moore  e t a l , 1976, and M c N a l l y ,  1 9 7 6 . L i k e most s u r v i v a l d a t a i n  the  the data  and McNally  literature  s u r v i v a l measurements  of  both  at survival  Moore  l e v e l s under  0.5.  were  obtained  from  A l l of the results are  84  0.0  Figure  20:  2.0 o o s SURVIVAL CURVES,  4.0 <z c s "Y*  6.0 •>  EXPERIMENT  S E T B.  The concentration of misonidazole f o r the data points and s u r v i v a l curves 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, a n d 5- 5 . 0 mM misonidazole. The d r u g was present 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 l o w e r g r a p h i s a m a g n i f i e d v i e w o f t h e d a t a shown i n t h e u p p e r graph in, the r e g i o n 0 - 7 . 5 Gray.  85  TABLE V  Table V:  DMF OF 0 - 5 . 0 mM MISONIDAZOLE EXPERIMENT SET B .  AT SURVIVAL L E V E L S OF 0 . 0 1 TO 0 . 8 ,  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 a s 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 experiment 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 a s 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 b y 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 . T h e 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 intervals. R e s u l t s f r o m M o o r e e t a l (1976) a n d M c N a l l y (1976) a r e l i s t e d for comparison.  [MISO] (mM)  DMF a t S = 0 . 8  DMF a t  S=0.5  DMF a t S = 0 . 1  DMF a t  S=0.01  FROM EXPERIMENT SET B : 0.1  1.0±0.4  1 . 0±0.4  1.010.4  1. 110.3  0.4  1.1±0.4  1 . 2±0.4 -  1.310.4  1. 410.4  1.5  1.3±0.4  1 . 5±0.3  1.810.3  1. 910.3  5.0  1.4±0.4  1. 710.4  2.110.4  2. 310.3  0.1  1.2  1. 3  1.0  1.8  1. 8  15  2.0  2. 3  2.1  2.3  FROM MOORE e t a l  FROM MCNALLY 5.0  (1976):  (1976):  86  consistent. equation  Thus  to  the  misonidazole  through 0.01  et  a  figures  (1982)  have  a procedure  to  about  1 and 2.  the misonidazole been  clearly  this  study.  Part of  recently  set B gives  similar  to  at  the that  survival  of  0-5.0  the motivation  for  result  the  set  was  radiosensitization  is  t h e most  accurate r e s u l t s .  beta  0 . 1 mM m i s o n i d a z o l e .  to  to  0.4  It  approximately  misonidazole  can  at  for  of  the  not  be  OER  this  of  levels.  the  many  to  (Adams,  1973;  results.  The  concentrations  are  inactivated,  and  only  10% b y  can  radiotherapy  i n f r a c t i o n a t e d r e g i m e s where make  then  the  dose  Wardman, data  one  drug  can  of  experiment  the  parameter  concentration  range  0 mM a n d  survival  is  mM a n d o n l y b e t a  is  greater of  than  2  90%  Gy  in  by  the  or  tumour  significantly  f r a c t i o n s are s m a l l . could  equal  hypoxia,  enhance  effective  to  1977).  beta.  not  in of  analagous  coefficient  significantly  by  to  term,  not  the  quadratic  inactivation  fractions  be  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  the  used  s i g n i f i c a n t l y between  0-0.1  now  mechanism  15 shows t h a t  cells  alpha  T h i s has  conditions  since  c o n c e n t r a t i o n range  at  if  by  0.7  2.8  s t u d y was t o d e t e r m i n e  all  the  from  found  in  of  misonidazole  They  a s shown  at  20%  asynchronous  here.  than  survival  Figure  for  decreases  that  drug  dose  the  calculated  alpha,  larger  reported  experimental  change  misonidazole  coefficient like  does  Therefore  increase  mM.  OER  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  mM a n d t h a t  observed  concentration  one  considered  s i g n i f i c a n c e of  i n the  linear-quadratic  high  the  anticipated  Clinical  observed  at  the  levels greater  DMF a l s o d e c r e a s e s a t h i g h  This  a l p h a does not  fit  measured  the mechanism of oxygen r a d i o s e n s i t i z a t i o n 4.3.2  to  values  analysis  1.7  demonstrated  misonidazole  increase  survival  model-independent  survival  not  mM).  al  using  need  measured  (5-15  Palcic CHO c e l l s  alpha  linear If  a  cell improve  The u s e  increase  of the  87  radiosensitization,  provided  other  comparison  respects  employed. figure  These o b s e r v a t i o n s 20.  measurements than  It  is  and  the  approximately  due t o t h e by  in  such  to  a  best  fit  3 Gray.  a regime  regime  to  not  where  survival  curves may  a disadvantage  smaller  in  fractions  are  s u r v i v a l c u r v e s shown  distinguish  Misonidazole  c y t o t o x i c i t y of  is  are r e f l e c t e d i n the  impossible  between  at  doses  still  of  provide  t h e d r u g and t h e enhancement  the  of  in  survival  radiation  less  a clinical  gain  the c y t o t o x i c i t y  radiation. The d o s e m o d i f y i n g  the  data  of  high  as  30 G r a y ,  for  0.4  as about  of  have  measured  only  at  0.01 the  survival  tabulated  mM f o r  3 Gray,  4.3.3  levels  The  experimental clinically  DMF o f  error,  doses  less  than  the  This to  a  as  dose  0.5.  of  the  12  shows  radiosensitizer  this  clearly fails  Radiobiological test  reasonable  fit  (table to  the  III)  the  survival  s i g n i f i c a n t e f f e c t on t h e for  significance.  to  Gy.  misonidazole  Other  f r o m two to  not  1.5  as  mM  for  as about  2 at  researchers system  but  investigators  within  decrease c e l l  they  are  reasonable survival  at  concentrations.  model  Furthermore,  concentrations  is  doses  show t h a t  According to  a l p h a component  for  5 . 0 mM m i s o n i d a z o l e  that,  linear-quadratic data.  for  same e x p e r i m e n t a l  study  DMF  for  doses as h i g h  The r e s u l t s of  The  12 G r a y ,  occurs for  in  IV.  mM m i s o n i d a z o l e  5 . 0 mM f o r  results  experiment  0.1  table  as h i g h  and f o r  2.110.5.  in  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  chi-square  increase  for  misonidazole  with  the  o f m i s o n i d a z o l e has been e s t i m a t e d  tabulated  unity  corresponding  along  consistent.  (DMF)  B and  from  The maximum DMF i s  survival  are  set  different  doses as h i g h Gray.  factor  experiment  significantly  a  that  the  results  (equation  3)  the is  a  has  no  and c a u s e s t h e b e t a component  to  of  0.4  misonidazole  of  mM  and  greater.  The  88  molecular theory that  alpha  multiple  is  cell one  then  hit  important  If  not  to  acknowledge (if  only  more  that  these  dose  bias  in  This  bias  and  The  choice of  equation  analysis  after of  on c e l l  easier  to  method  viable  assays. cells  irradiation.  the  the  the beta  component  of will  the  time  course  to  cells,  X-irradiation.  etc.)  This  3 to it  fit  is  data  does  s i m p l y one o f fit  the  as  the  data  on r a d i a t i o n  and dose  the  by a  The  set B resulted  data.  The  cell  survival  in  following  source of day  for  measured  by  the  discrepency  is  the  the  low  locating cells prediction  cells  method.  experiments:  macroscopic  after  the  experimental  obtain  location  is  fit  i n the l i t e r a t u r e ,  experiment  future  reduced  of  of  The m a j o r  on  c a n be  survival.  c o r r e l a t i o n between  significantly  shrivelled  exposed to  theory;  experimental procedure  conventional  c a n be  is  this result  which can adequately  improvements  and  the  selecting  hours  affects  s u r v i v a l measurements  s u r v i v a l models  s h o u l d be c o n s i d e r e d f o r  Improve  low  cells,  the  accurate  1.  measurably  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  to  proposes  and b e t a  i n t o the s i g n i f i c a n c e of  not most)  3.1.  Suggested  improvements  24  study  the e f f e c t of misonidazole  improvements  inactivation  i n t e r a c t only w i t h the m u l t i p l e h i t  complex m a t h e m a t i c a l e q u a t i o n s  4.3.4  (1981)  research.  section  c a n be r e a d i l y  a  cell  c o n s t i t u t e a b i a s toward the molecular  least  of  of  misonidazole  Further  e q u a l l y w e l l by s e v e r a l in  Chadwick and L e e n h o u t s  component  i t may o n l y  to future  discussed  s u r v i v a l of  component.  inactivation.  be l e f t It  the  hit  component cell  is  of  events  treated  dose  during  based  on  (formation with  assay.  the  first  qualitative of  misonidazole  giant are  89  2.  Use t h e  Otherwise,  it  low dose assay o n l y  i s e a s i e r t o measure  t h e same d e g r e e 3.  Choose  of dose  suited to  fits  important  as the  to  points  can  models.  least three  Note  for  of  be  sorter  increased which  plating  to  increase  the  approach  plates  efficiency  permits,  the  cells if  after  less  reliability  the  that  of  that  the  to  the  obtain  task  of  requiring  the  Another  procedure assay and,  computer  would  time  method  locating cells  t o teach the  0.5.  high  for  is to  have  in addition,  all  to  of  the  is  low  dose  them.  counted  number  of  to  of  as  cells  well  are  each r a d i a t i o n  the  as  dose  estimate cells  cells  conventional  assay  by  This  will  cells  and  plated.  location the  using  of in  of  to  the  from d i g i t i z e d  statistical  the  advantages  assay a  cell  increase it  If  will  cells  computer)  low dose  of  the also  plated  cells  experimenter  a d d a TV d i g i t i z e r  the  to  parameters.  (perhaps a u t o m a t i c a l l y by  relieving  recognize  as  dose p o i n t s  Use s y n c h r o n o u s  r e l i a b i l i t y of  consuming  or  way  Such d a t a i s  the c o n v e n t i o n a l assay c o u l d approach the r e l i a b i l i t y of  microscope.  than  experiments.  are then c l a s s i f i e d w i t h the microscope  without  a  a reasonable  survival.  counting  debris  such  e s t i m a t i n g s u r v i v a l model  in order  deviation  in  the s u r v i v a l .  s u r v i v a l measurements  concentration  When t e c h n o l o g y  estimates  logarithm of  t o reduce the v a r i a t i o n between 5.  greater  s u r v i v a l with the conventional assay  survival  low dose p o i n t s  t h e mean a n d s t a n d a r d order  for  i n the  survival  Obtain at  and m i s o n i d a z o l e  survivals  accuracy.  o b t a i n even increments  4.  t o measure  assay  under the  the  tedious  ALDAS c o m p u t e r TV p i c t u r e s . of  w o u l d b e much f a s t e r t h a n t h e c u r r e n t  the  low  and This dose  procedure.  90  4.4  SURVIVAL MEASUREMENTS AT LOW DOSES. The  low dose  assay was used  capacity  f o r survivals  automated  low dose assay system  strain  resulting  of greater  in a  ten c e l l s  than  locating  cells  proliferative cells.  The  increase  i n productivity  and  U s i n g ALDAS, t h e experimenter can l o c a t e  o r 150 c e l l s  assume a p l a t i n g o f about 0.2 c e l l s / m m when  of c e l l  50% o f t h e p l a t e d  two- t o t h r e e - f o l d  p e r minute  loss  (ALDAS) reduced assay time and experimenter  reduced e x p e r i m e n t a l u n c e r t a i n t y . about  t o measure  two t o t h r e e  i n 15 minutes.  These  figures  and a f i e l d o f view o f about 1 mm  2  2  days  after  irradiation.  Lower  plating  d e n s i t i e s , a s m a l l e r f i e l d o f 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 t o d e c r e a s e t h e speed w i t h which  cells  can be l o c a t e d .  can  hours  p e r day, (approximately 16 p l a t e d  locate  vessels,  cells  f o r up t o f o u r  locating  b e f o r e becoming  150 c e l l s  consecutive  days  T h i s c o u l d be g r e a t l y improved by f u r t h e r automating ALDAS ( s e c t i o n  4.3.4).  listed  found  that  t h e system  significant  three  errors.  has been  and making  f o r about  observational  It  overstressed  per vessel)  The experimenter  consistently  meets  the s p e c i f i c a t i o n s  i n table VI.  The  interactive  F o r t r a n programs  necessary  and s u f f i c i e n t  selected  alternatives  flexibility  options,  to  and ease o f use.  give  developed f o r t h e LDA system p r o v i d e  variable the  program  optimum  The programs  parameters,  balance  are w e l l  between  and u s e r software  documented and can be  e a s i l y updated o r m o d i f i e d f o r u n f o r s e e n a p p l i c a t i o n s . Some r e a d e r s may be i n t e r e s t e d i n a c q u i r i n g t h e r e own ALDAS. of these s p e c i f i c a t i o n s  i s currently  A system  n o t s t o c k e d by any manufacturer.  b u i l d and t e s t t h e system r e q u i r e s t h e s e r v i c e o f one computer  To  technician  91  TABLE  Table VI:  SPECIFICATIONS  Step  size:  Scan  dimensions:  FOR THE AUTOMATED LOW DOSE A S S A Y , A L D A S :  10 um 7 cm X 12 cm  Repeatability:  100 um  Minimum  l e s s than  repeatability:  approached Maximum s p e e d , A S C A N :  VI  10 um i f from the  l o c a t i o n s are  always  same d i r e c t i o n .  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 . s p e e d s may g e t m i s s e d s t e p s a t e d g e o f the load i s too  Maximum s p e e d , MSCAN:  s p e e d s may g e t m i s s e d s t e p s a t e d g e o f load i s too  higher  scan or  if  heavy.)  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 .  the  (At  heavy.)  (At  higher  scan or  if  92  (to b u i l d  the  machinist  (to  months.  It  i n t e r f a c e between the build would  become p r o f i c i e n t  stage) ,  take  about  the  and  two  at using the  Research i n t o survival  the  and the  a computer to  four  stepping motors),  programmer  weeks  for  for  an  one  one  to  two  experimenter  to  l o w dose a s s a y a l o n g w i t h ALDAS.  effects  of  s h o u l d be e n c o u r a g e d .  radiosensitization,  computer  low doses  The  exemplify  the  of  results need  ionizing  of  for  this  radiation  study  direct  for  on  cell  misonidazole  measurements  at  low  The  low  doses.  4.5  FUTURE RESEARCH 4.5.1  Discrepency  in  survival  measurements  dose assay tended t o y i e l d s l i g h t l y h i g h e r dose is  assay  as  noted  0.12+0.11  These  for  in  section 4.2.3.  experiment  comparisons  were  set  made  at  A a  between  values of  The  and  average  0.18±0.12  survival  s u r v i v a l than the difference for  level  developement  plated and More  cells  were  and  detailed  cell  into  non-viable  counted  in  research  the is  a  colony;  within  two  low dose required  non-viable  in  the say  how t h i s  experiment day a f t e r  could  relative one-half  relative yield  irradiation  more q u a n t i t a t i v e  a  number day  of  about  measure  of  of  the  is,  three when  days the  clearly  cells  from the on t h e  factor are  maximum  that  for  time  after  the  become time  B.  and  can  of  on the  irradiation  were  demonstrate  located.  this;  low dose  for assay  macroscopically  of  irradiation  resulting  It  set  and X - r a d i a t i o n  cells  survival  located.  50%  high  survival  a proportion  cells plated for  number d e p e n d s  i n which c e l l s  to  to  intervals  correction  that  assay  one c o u l d f o l l o w t h e g r o w t h o f  determine  measure  a  became  not  example,  of  in  experiment  q u a l i t a t i v e l y be e x p l a i n e d b y t h e e f f e c t o f m i s o n i d a z o l e the  assays.  survival. depending  and This  on  could also provide  which should  elapse  the a  between  93  cell  irradiation 4.5.2  and c e l l  Survival  location.  dependence  on  oxygen  tension.  This  experiment  has  3 shown t h a t decrease  for  in  an oxygen t e n s i o n o f  cell  survival  presence of  0 - 5 mM o f  to  these  perform  experimental present  low  measurements  low  ppm  doses  of  (0.1%)  there  at  used i n  doses  of  other  this  oxygen  to  It  measurable  Gray)  in  may be o f in  may be  mask  no  (0-2  tensions  thesis there  irradiation  is  irradiation  the r a d i o s e n s i t i z e r misonidazole.  conditions  at  at  10  that  value  for  sufficient  the  the  the  oxygen  effect  of  the  The  low  dose  radiosensitizer. 4.5.3 assay  Observation  with  ALDAS  X-irradiated the  of  r a d i a t i o n dose,  the  interaction  our  own l a b  cellular ideal  cells.  Patterns  of  et a l ,  4.5.4  of  and  for of  cells  division.  studying  growth which  the  could  be  follow  this  type  radiation  1981a and  would  with  lead  cells.  labs.  to  a refined  Such work  has  (Colombo and M a r i n ,  presence agents, clear  low  (or the  how  absence)  location of measurements  extrapolated doses.  doses.  to  These of the  at  low doses of  include  radiosensitizers, cells  it  The r e s u l t s c o u l d h a v e  some l i g h t  effects  obtained  lower doses  described  and  patterns  on  f a c t o r s c o u l d be understanding  of  already  in  begun  Sakka e t  al,  1981b.).  S u r v i v a l measurements  at  of  1963;  ionizing  s u r v i v a l e f f e c t s w h i c h have been measured a t h i g h doses of measured  progress  these  drug concentration, or other environmental  as w e l l as o t h e r  Grote  growth  system  proportion  Research of  the  1975;  an  mammalian  dependenc  measured.  is  of  i n the at  cell  higher  w o u l d be u s e f u l important  on b a s i c r a d i o b i o l o g i c a l  the  radiation  effects  radioprotectors cycle,  etc.  doses  of  of  Cell can be  LET, and  Since i t radiation  t o make m e a s u r e m e n t s  clinical  questions.  radiation.  the other  is  not  can  be  at  low  i m p l i c a t i o n s a n d may  shed  94  4.6  SUMMARY. The p u r p o s e  of  misonidazole  of  this  s t u d y was t o m e a s u r e  at  low  doses  necessary  to  assay  locating  by  following  their  uncertainty more.  in  location system  of  and  automated five  experiment  The high  and  parameter,  the  built  accurate  conventional  at  to  were  than  of  improved  in  one  the  this  improvement  the  levels  3 was  for  ease  obtained  obtained  required  to  varied  or  cell  using  this  assess the  effect  the  control  Repetition  the  significantly  in  With  uncertainty  to  50%  manually.  (including  experiment.  fit  of  stepping-motor-driven  due t o v a r i a t i o n s b e t w e e n  equation  survival and  easily  survival  cell  was  irradiation  provide  misonidazole  this  after  survival  results  t h e measurements  beta,  it  With  3-10%  Results  and r e d u c i n g t h e e r r o r s  dose  accomplish  immediately  to  c o u l d b e made  linear-quadratic  ability  computer-controlled  concentrations  greatly  radiosensitizing  To  microscope.  a  designed  system a l l of  different  statistics  a  was r e d u c e d  much more  w i t h no m i s o n i d a z o l e ) same  using  classification.  were  of  cells  incorporating was  radiation.  uncertainty  individual  survival  stage and  the  growth  A system  microscope  the  improve  of  the  be  the  improving  experiments.  survival in  of  the  data.  The  misonidazole -2  c o n c e n t r a t i o n range The Thus  low  dose  (low)  to  parameter,  although  radiation,  0.1  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 alpha,  misonidazole  these  doses  of  misonidazole  in  results  patient  value of measuring  cell  constant  radiosensitizes  show  radiation.  remained  a  This  treatments  minimal may to  cells  at  0.03910.010 to  high  radiosensitization  explain date.  s u r v i v a l at low doses of  the  This  minimal study  radiation.  . Gy  doses at  \ of  clinical  success  exemplifies  of the  95  BIBLIOGRAPHY  A d a m s , G . E . (1973) Chemical r a d i o s e n s i t i z a t i o n of hypoxic B r i t i s h M e d i c a l B u l l e t i n , 29, 48.  cells.  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 low doses of 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 : proceedings of the s i x t h L.H.Gray Memorial Conference held at Bedford C o l l e g e , London, 16-21 September, 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 Radiobiology. Cambridge 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. (1976). Electron affinic sensitization. VII: A c o r r e l a t i o n between 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 hypoxic 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 Research, 67, 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 . , Ings, R., and M c F a d z e a n , J . A . (1974) Metronidazole ( " F l a g y l " ) . A r a d i o s e n s i t i z e r of hypoxic B r i t i s h J o u r n a l of R a d i o l o g y , 47, 474. Bacq,  cells.  Z . M . , a n d A l e x a n d e r , P . (1961) Fundamental of Radiobiology. Pergamon P r e s s .  B e g g , A . C . , S h e l d o n , P . W . , a n d F o s t e r , J . L . (1974) Demonstration of r a d i o s e n s i t i z a t i o n of hypoxic metronidazole. B r i t i s h J o u r n a l of R a d i o l o g y , 47, 399. B e n d e r , M . A . , a n d 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 m a m m a l i a n I n t e r n a t i o n a l Journal of Radiation Biology, Bevington, 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 McGraw-Hill, Inc., U.S.A.  for  cells  cells in 5, 133.  the p h y s i c a l  i n s o l i d tumors  vitro.  sciences.  by  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 Masson P u b l i s h i n g U . S . A . , Incorporated.  Management o f  Cancer.  B r o s i n g , J . W . (1983) M e a s u r e m e n t s o f C e l l S u r i v i v a l a t Low D o s e s 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 Columbia, Canada. C h a d w i c k , J . D . , a n d 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 . Springer-Verlag. Chapman, C . J . , a n d 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 events and t h e i r time s c a l e A d v a n c e m e n t s 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 . , a n d 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 of c o l o n i e s developed i r r a d i a t e d iri v i t r o with X-rays. E x p e r i m e n t a l C e l l R e s e a r c h , 29, 268.  i n mammalian  f r o m mammalian  cells.  cells  Denekamp, J . , a n d 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 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 Research, 61, 191. Denekamp, J . , M i c h a e l , B . D . , a n d 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 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 _ i n v i v o . R a d i a t i o n R e s e a r c h , 60, 119. D i s c h e , S . (1978) Hypoxic c e l l s e n s i t i z e r s i n radiotherapy. I n t e r n a t i o n a l Journal of Radiation Oncology, 157.  Biology,  electron  a n d P h y s i c s , 4^,  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 . In: Radiation S e n s i t i z e r s : T h e i r U s e i n t h e C l i n i c a l Management o f Cancer. 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 . 349.  of  97  D u r a n d , R . E . a n d 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 v i t r o tumor m o d e l . In: Radiation Sensitizers: T h e i r Use i n t h e C l i n i c a l Management Cancer. Masson P u b l i s h i n g U . S . A . , I n c . 152. E l k i n d , M . M . , a n d S u t t o n , H . (1959) X - r a y damage a n d r e c o v e r y i n m a m m a l i a n c e l l s N a t u r e , 184, 1293. 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 M a m m a l i a n G o r d o n a n d B r e a c h , New Y o r k .  in  in of  culture.  Cells.  F a d d e g o n , B . A . (1982) Documentation o f the s o f t w a r e o f the automated low dose assay system (ALDAS). B i o p h y s i c department, B r i t i s h Columbia Cancer Research Centre, Vancouver, B r i t i s h Columbia, (unpublished) 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 , 46, 234. F r i c h e , H . , a n d H a r t , E . J . (1966) Chemical Dosimetry. I n : R a d i a t i o n D o s i m e t r y , volume Roesch, W.C., ed. A c a d e m i c P r e s s , New Y o r k . 1 2 6 .  II,  Instrumentation.  Attix, F.H.,  and  G r o t e , S . , J o s h i , G . , a n d 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 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. Observation of r a d i a t i o n - i n d u c e d mammalian c e l l s i n c u l t u r e , and I n t e r n a t i o n J o u r n a l of R a d i a t i o n Hall,  (1981b) chromosome f r a g m e n t l o s s i n i t s e f f e c t on c o l o n y - f o r m i n g B i o l o g y , 3 9 ( 4 ) , 395  E . (1973) Radiobiology for the R a d i o l o g i s t . H a r p e r a n d Row, New Y o r k .  live ability.  the  98  Heye,  R . R . , K i e b l e r , E . W . , A r n z e n , R . J . , a n d 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 photomicrography of 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 . Report 16, L i n e a r Energy T r a n s f e r . J a k o b y , W . B . , a n d P a s t o n , I . H . , e d . (1979) Methods i n Enzymology, Volume L V I I I : C e l l A c a d e m i c P r e s s , New Y o r k .  Culture.  K e l l e r e r , A . M . , a n d 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 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 . , a n d R o s s i , H . H . (1978) Generalized formulation of dual radiation R a d i a t i o n Research, 75(3), 471.  (1970)  functions.  action.  K e m p e r , 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 guide. R a d i o b i o l o g y Research D i v i s i o n , Department of R a d i o l o g y , Standford U n i v e r s i t y School of Medicine, Standford, C a l i f o r n i a , (unpublished) 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) Radiation-enhanced c y t o t o x i c i t y of misonidazole. R a d i a t i o n R e s e a r c h , 88, 343. M c N a l l y , N . (1976) The e f f e c t o f a c h a n g e 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 a f f i n i c s e n s i t i z e r s to sensitize hypoxic 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 of R a d i a t i o n B i o l o g y , 29, 191. 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 Ro-07-0582 i n h y p o x i c cells. 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 , Canada. Moore, 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 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 , 67, 459.  electron  mammalian  Ro-07-0582  99  O k a d a , S . (1970) Radiation Biochemistry. 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 a n d DNA damge i n h y p o x i c m a m m a l i a n B r i t i s h J o u r n a l o f Cancer, 37, Supplement I I I , 54. 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 B r i t i s h J o u r n a l of Cancer, 46, 980. P a r k e r , L . , S k a r s g a r d , L . D . , a n d 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 mammalian c e l l s t o X r a y s by N-oxyl. 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 Research, 38, 493.  cells.  ratio.  triacetoneamine  P u c k , T . T , a n d M a r c u s , P . L . (1956) A c t i o n o f X - r a y s on mammalian c e l l s . J o u r n a l of Experimental Medicine, 103, 653. S a k k a , M . , K a t s u t a , H . , a n d 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 mamalian c e l l s irradiation. 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.  after  S i n c l a i r , W . K . (1966) The s h a p e 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 m a m m a l i a n in vitro. In: Biophysical Aspects of Radiation Quality. IAEA, V i e n n a , 21.  gamma  cells  T h o m s o n , J . E . , a n d R a u t h , A . M . (1974) A comparison o f t h e e f f e c t i v e n e s s o f NF-167 and m e t r o n i d a z o l e c e l l s e n s i t i z e r s o f KHT t u m o r c e l l s i n v i t r o . R a d i a t i o n Research, 60, 489. 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 , C l i n i c a l t e s t i n g of the r a d i o s e n s i t i z e r Ro-07-0582. tumours. C l i n i c a l R a d i o l o g y , 27, 167.  cultured  as hypoxic  L . M . (1976) I l l - Response  of  100  T h o m l i n s o n , R . H . , a n d 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 a n d t h e implications for radiotherapy. B r i t i s h J o u r n a l o f C a n c e r , j), 539.  possible  T o b i a s , C . A . , B l a k e l y , E . A . , N g o , F . Q . H . a n d 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 Withers, H.R., ed. R a v e n 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 . , a n d 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 . , a n d F r y e r , C . G . (1977) C l i n i c a l phase I study of the hypoxic c e l l r a d i o s e n s i t i z e r Ro-07-0582, 2-nitroimidazole derivative. R a d i o l o g y , 122, 801. Wardman, P . (1977) The u s e o f n i t r o a r o m a t i c compunds a s 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 Research Q u a r t e r l y , 1 1 , 347.  a  101  APPENDIX  L i s t i n g s of  t h e ALDAS  routines.  The a u t o m a t e d l o w d o s e a s s a y routines, OUT50,  (ALDAS)  software c o n s i s t s of  LDALOC a n d L D A C L A S S , a n d f i v e  and P L I N E .  (See s e c t i o n 2 . 4 ) .  subroutines,  two  MSCAN, A S C A N , B E E P ,  This appendix contains a l i s t of  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 b y l i s t i n g s o f routines OUT50,  a n d t h e t w o s u b r o u t i n e s MSCAN a n d A C A N .  and PLINE i n v o l v e  simple input  major  and o u t p u t .  the  the  major  The s u b r o u t i n e s B E E P , These are not  listed  here.  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  Subroutines  Variable  Type  C (IDUM)  Integer array  -  number o f t i m e s c l a s s i f i c a t i o n IDUM o c c u r s i n LDA d a t a f i l e .  CLASS(NLOC)  "  -  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,  CLASS(ICLASS,NLOC)  "  -  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.  Integer  -  # o f s t e p s t o b e m o v e d / a c t u a l l y moved b y X motor.  DX  Comment  LDALOC.  DXH  - max. # o f motor.  s t e p s t o be moved CW b y X  DXL  - max. # o f motor.  s t e p s t o b e moved CCW b y X  DY  # o f s t e p s t o b e m o v e d / a c t u a l l y moved b y Y motor.  DYH  - max. # o f motor.  s t e p s t o b e moved CW b y  DYL  - max. # o f motor.  s t e p s t o b e moved CCW b y  Y  Y  102  APPENDIX  (continued)  Variable  Type  Comment  FNBAK(I)  Byte  -  array containing filename of f i l e 9.  Fortran  FNDAT(I)  -  array containing filename of f i l e 7.  Fortran  FNLOC(II)  -  array containing filename of f i l e 6.  Fortran  FNPRN(I)  -  array containing filename of f i l e 8.  Fortran  -  c l a s s i f i c a t i o n s e t i n L D A C L A S S , LDAFREQ.  IDUM  -  m u l t i p l e use  JVAL  -  value of b i t s  -  l o c a t i o n s 1 / P b y u s e r f r o m LDALOC u s e d by LDACLASS.  -  index of  NLOCM  -  # of v a r i a b l e s stored i n array  NSTEP  -  index of  NSTEPM  -  # 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 LDAREF a n d u s e d b y LDALOC.  -  scan 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  ICLASS  LOC(I,NLOC) NLOC  SCAN(I,NSTEP)  SDIR XH  Integer  Integer  array  Integer  Integer  array  Byte Integer  variable.  array  1/P  from j o y s t i c k . and  LOC. LOC.  a r r a y SCAN.  - max. v a l u e o f  XSTEP p e r m i t t e d .  XL  -  min.  XSTEP p e r m i t t e d .  XREF  -  reference position, X direction.  XSTEP  -  p o s i t i o n of stage along X a x i s side) i n # of steps.  YH  -  max. v a l u e o f  YL  -  min.  YREF  -  reference p o s i t i o n , Y d i r e c t i o n .  value of  YSTEP p e r m i t t e d .  v a l u e o f YSTEP p e r m i t t e d .  from  scan.  (side-to-  103  APPENDIX  (continued)  Variable  Type  Comment  YSTEP  Byte  - p o s i t i o n of stage along Y a x i s back) i n # of 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 m o t o r s a n d p o r t 25 f o r t h e speakers.  CDTNX  -  no X m o t o r m o t i o n i f  CDTNX =  0.  CDTNY  -  no Y m o t o r m o t i o n i f  CDTNY =  0.  (front-to-  stepping the joystick  I END  Integer  -  # o f beeps 0 / P by s u b r o u t i n e  KIND  Byte  -  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 b y user through j o y s t i c k .  Integer  -  # o f s t e p s t o output t o X motor 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. # of steps to output to X motor, s u b r o u t i n e ASCAN.  NX  -  -  NY  -  BEEP.  # of s t e p s t o output t o Y motor 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. # of steps to output to Y motor, 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 DO LOOP c l o c k .  -  0 - no m o t i o n , 1-CW m o t i o n , motion, X motor.  YBIT  -  0 - no m o t i o n , 1-CCW m o t i o n , motion, Y motor.  XDIR  -  - 1 - C C W m o t i o n , 1-CW m o t i o n , motion, X motor.  0 -  no  YDIR  -  - 1 - C C W m o t i o n , 1-CW m o t i o n , motion, Y motor.  0 -  no  -  d i g i t a l value of of j o y s t i c k .  X (side-to-side)  -  d i g i t a l value of of j o y s t i c k .  Y (back-to-front)  SPEED  XBIT  XJOY  YJOY  Byte  Integer  speed  through  2-CCW  2-CW  axis  axis  LDALOC  cccccc  FOR  PAGE 01  CCCCCC L o c a t i o n scan f o r low dote a s s a y . CCCCCC BYTE OPTION.KIND.SDIR.LINE( 134) , NL F NE .PAGE (AO) BYTE F N L O C d l > .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 c o u r . t i r . s M ! ') 1200 FORMAT!/' LOCATION SCAN MENU. ( E n t e r o p t i o n f r o a c o n s o l s ) ' / 1/' A: S t a r t r e a u l a r s c a n . '/ 2' B: S t a r t d i s c r e t e p o i n t s c a n . ') 1201 FORMATC 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 . ' . 1' and scar, p o i n t s i f any.'/IX.'D! R e d e f i n e s c a n ' . 2' p a r a m e t e r s . ' / I X * ' E ! S u b a i t p r i n t o u t t o p r i n t e r . ' / l X . 6'Z! E x i t r o u t i n e . ' / ) 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 s p e e d . 10 = a a x i a u a . ' / l X . 2'B! CELL COUNT'»9X.'1-40' . IS.' Max. no. o f l o c a t i o n s ! 1 0 ' . 3' t o be r e c o r d e d . ' / I X . ' C I COINCIDENCE CHECK 0-1'. 415.' 0 * A u t o a a t i c c o i n c i d e n c e check.'/34X. 5'l»Coincidence check f r o a MANUAL SCAN'/34X'ALTERNATIVES. '/ 4' D! COINCIDENCE RANGE 0-127'.15.' D i s t a n c e i n which t o ' 7/34X.'search f o r c o i n c i d e n c e s . ' / B' SCAN PATTERN PARAMETERS!'/' E ! LINE SPACING'* 94X. '1 - 127'.15.' Nuaber o f s t e r s * 1 0 between scan l i n e s . ' / . 1- F ! POINT SPACING' .SX.' 1-127' . 15. ' Nu.be r o f s t e p s a l O '. 2'between scan p o i n t s ' / 3 4 X . ' f o r d i s c r e t e p o i n t s c a n . ' / 3' G! DATA FILE'.1IX.'0-9'.15.' Nuaber. n. d e f i n i n g d a t a ' . 4' f i l e f o r 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 ' . I D 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 beepi n p u t from J o y s t i c k b u t t o n s . ' /  LDALOC  FOR  PAGE 02  3 ' Two b e e p s J o y s t i c k c o n t r o l s scan speed.'/ 4' Three beepsJ 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' F i v e beepsl 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 f o u n d . 2.3.4-Exit r o u t i n e . ' . 2 ' (No d a t a o u t p u t ) ' ) 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 w i t h ' . 2 ' c l a s s i f i c a t i o n . '/5X» ' 3 - C o i n c i d e n c e c h e c k . '»5X» 3 '4-Resuae s c a n . ' ) 5020 FORMAT(' AUTOMATIC SCAN ALTERNATIVES! '/ 1 5X.'1.2-Go t o M a n u a l s c a n . 3-Delete last recorded'. 2 ' l o c a t i o n . ' /5X *' 4-Return s t a a e t o r e f . 3 'erence p o s i t i o n and e x i t * ' ) 5025 FORMATC DISCRETE POINT SCAN ALTERNATIVES! '/ 1 5X.'l-Record l o c a t i o n . 2-Go t o M a n u a l s c a n . ' / 2 5x*'3-Resuae s c a n . 4-Return s t a a e t o 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! E n t e r u s i n s J o y s t i c k b u t t o n s . ' ) 5040 FORMAT(' POSSIBLE COINCIDENCE. ALTERNATIVES!'/ 1 5Xr'1.2.3.4-Move s t a s e t o l o c a t i o n o f c o i n c i d e n c e . ' ) 5045 FORMAT(' COINCIDENCE CHECK COMPLETED•') 5060 FORMAT ( ' HANG IN THERE T I L I GET THIS DATA OUT....') 50B0 FORMATC Movina s t a a e t o r e f . p o s i t i o n . Watch your hands*'/ 1 ' (YOU c a n s t o p i t w i t h t h e J o y s t i c k o r the b u t t o n s ) ' ) 5100 FORMAT(' THIS IS THE REFERENCE SCAN FOR THE LOW DOSE ASSAY.'/ 1SX'FANILIARIZE YOURSELF UITH THESE SIGNALS!'/ 25X»'0ne beepinput f r o . J o y s t i c k buttons.'/ 35X.'Two b e e p s J o y s t i c k c o n t r o l s s c a n speed.'/ 45X,'Three b e e p s 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.'/ S S X ' F i v e beepsa l l done s i s n s l . ' / / 6' SCAN LOCATIONS ARE STORED IN F I L E •'.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 S X . ' l - R e f . p o s i t i o n found. 2,3.4-Exit routine.'. 2 ' (No d a t a o u t p u t ) ' ) S140 FORMAT(' LOCATE SCAN POINTS. OPTIONS! '/ 1 SX.'l-Record l o c a t i o n . 2.3-Return s t a a e t o r e f . 2 'erence p o s i t i o n and e x i t . ' / 3 5x.'4-Exit routine. ( S t a a e not M o v e d . ) ' ) 4200 FORMATC LOCATION SCAN FOR F I L E •',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 F I L E •'.8A1,'.',3A1. I ' ! ' / ' X L « ' . I 5 . ' XH""'.I5.' Y L - ' . I S . ' Y H » ' . I 5 . ' 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) C Set ADC O/p p o r t s 24 and 25. CALL 0UT(24.15> CALL 0UT(25>25> C Open f i l e 8 ( f o r printout). CALL 0PEN(8.FNPRN.2> C S e t proaram p a r a m e t e r s f o r d e f a u l t v a l u e s .  j  O | . '  ]  -  1 I | ;  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 L o c a t i o n 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 r e c o r d e d l o c a t i o n s ) . 10 «RITE(5.5000> READ(5»4010)(FNDAT(I), I*1»B> DO 105 I M . 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 o f s c a n r e f e r e n c e p o i n t s and automated s c a n p o i 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. G e n e r a t i o n o f scan p o i n t s f o r r e s u l e r and d i s c r e t e p o i n t scans 20 IDUrl«*F-ARrl(5)»I0 NSTEF'M=0  LDALOC  FOR  PAGE 04  DO 210 I > l t 2 0 0 . 2 SCAN<l.I>-XLtIDUMt((Itl)/2> S C A N ! 1 . I t l I'SCANU .I> NSTEPM-NSTEPHT2 210  IF(SCAN(1.I)TIDUM.GT.XH)G0T0  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 230 JDUM-SCAN(2,I > 107 ENDFILE 6 XSTEP«XREF YSTEP-YREF C Auto s c a n , p r o s r e m c h o o s e s d i r e c t i o n from t h e a r r a y SCAN. User C c o n t r o l s speed o f s c a n w i t h J o y s t i c k . C JUAL r e t u r n e d f r o * ASCAN p r o v i d e s A l t e r n a t i v e s 1. 113 IF(NL0C.GE.PARM(2)«10)G0TO 300 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) CALL PEEP(S) GOTO 250 110 IF(0PTI0N.EQ.45>G0T0 111 C Determination o f next p o i n t t o scan t o f a r d i s c r e t e p o i n t 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 XSTEPl-SCANU.NSTEP) YSTEP1-SCAN<2.NSTEP> S D I R — 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 b r a n c h on J U A L . d i s c r e t e p o i n t scan o p t i o n s . URITE(3,S025> CALL B E E P ( l ) 122 JUAL=INP<24> JUAL"JUAL-(JUAL/lo>«14 IF(JUAL.LT.0)JUAL-JVAL+16 I F ( J U A L . E 0 . 1 5 ) G 0 T 0 122 DO 12? 1=1.10000  1 ! ! I  I |  —' O |V-T1 I , !  I I  I ] i  LDALOC 129  FOR  PAGE OS  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» r e g u l a r scan o p t i o n s . 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 s c a n . C o n t r o l o f s p e e d and d i r e c t i o n u i t h J o y s t i c k . C JUAL r e t u r n e d froe, MSCAN p r o v i d e s 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 2 0 0  LDALOC  FOR  PAGE 06  IF(IDUM.EO.1)GOTO 200 C Record s t a s e 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 b e f o r e beep. DO 500 1-1,1000 500 CONTINUE URITE(3,3030> CALL D E E P ( l ) C C l a s s i f i c a t i o n a t 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 d a t a t o d a t a f i l e 7 and p r i n t o u t t o 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 H F N D A T ( I ) , 1 - 1 , 1 1 ) 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 t o 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 R e f e r e n c e and s c a n p o i n t l o c a t i o n s c a n f o r low dose a s s a y . CCCCCC C Open f i l e 6 ( f o r d a t a o u t p u t ) . 40 CALL 0PEN(6,FNL0C,1> C One boundary c o r n e r 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 O p p o s i t e boundary c o r n v e r 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  ccccc  PAGE 01  CCCCCC C l a s s i f i c a t i o n scan f o r low dose a s s a y . 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 o p t i o n f r o a c', 1 ' o n s o l e ) ' / / ' A! S t a r t scan.') 1201 FORMAT( ' C: C a l c u l a t e f r e o u e n c y o f c l a s s i f i c a t i o n . ' / 1' D: R e d e f i n e scan p a r a a e t e r s . ' ) 1202 FORMATC E.' Subniit p r i n t o u t t o 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 P r i n t o u t . ' / 3 S X , ' 1 - C l a s s ' . 3 ' i f i c a t i o n f r e o u e n c i e s o n l y . '/38X . ' 2=A11 p r i n t o u t . ' / I X . 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 t o c l e s s i f y.'/38X >'6"Fi r s t s e t w i t h no c l a s s i ' , 4'fications.'/' COLONY LOCATION CONSTRAINT PARAMETERS!'/IX. 7'D! CLASSIFICATION SET 0-5'.17.' 0«No c o n s t r a i n t . ' / 3 8 X . 8'l-5«Set c o n t a i n i n a c o n s t r a i n t . ' / ' E! CLASSIFICATION '. 9'VALUE 0-5'.17.' C l a s s o f l o c a t i o n s t o be scanned.'/ 11X.' CLASSIFICATION FREQUENCY CONSTRAINT PARAMETERS!'/ 21X.'F! CLASSIFICATION SET 0-5'.17.' 0«No C o n s t r a ' . 3'int.'/38X.'l-5«Set c o n t a i n i n a c o n s t r a i n t . ' / / ) 1305 FORMATC ENTER LETTER OF PARAMETER TO CHANGE! '. 1 ' ( E n t e r Z t o 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 ' . I D 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 F I L ' . 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 1 2 3 4 5 6 7  PAGE 02  ' DOSE ASSAY.'/ ' FAMILIARIZE YOURSELF UITH THESE SIGNALS'. ' / ' One beepinput froa j o y s t i c k buttons.'/ ' Two b e e p s J o y s t i c k c o n t r o l s scan speed.'/ ' Three b e e p s 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 ' F i v e beepsa l l done s i g n a l . ' / / ' 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 d a t a o u t p u t ) ' ) 5010 FORMATC MANUAL SCAN ALTERNATIVES! '/ 1 S X . ' l - C I a s s i f y . ' 1 3 X . ' 2 - R e s e t scan c o o r d i n a t e s . ' / 2 SX.'3,4-Resuae s c a n . ' ) 5020 FORMATC Use J o y s t i c k t o c o n t r o l s c a n t r e e d t o next '. 1 ' l o c a t i o n . ' / 5 X . ' P r e s s any b u t t o n t o stop scan.') 5030 FORMATC CLASSIFICATION! E n t e r u s i n a J o y s t i c k b u t t o n s . ' ) S04O FORMAT(' CLASSIFICATION SCAN ALTERNATIVES.'/5x« 1 '1-Clessify.'13x.'2-Move '. 2 'staae.'/5X.'3-Resuae Scan.'.1 O x . ' 4 - E x i t r o u t i n e . ' ) 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 s t a a e t o r e f . p o s i t i o n * Watch your hands!'/ 1' (YOU c a n s t o p i t w i t h t h e J o y s t i c k o r any o f the b u t t o n s ) ' ) 5100 FORMAT(' THIS IS THE CLASSIFICATION FREQUENCY PROGRAM OF'. 1 ' THE LOU DOSE ASSAY.'/ 2 ' T h i s r o u t i n e c a l c u l a t e s the nuaber o f t i a e s each c l a ' * 3 ' s s i f i c a t i o n o c c u r e d '/ 4 ' f o r each s e t o f c l a s s i f i c a t i o n s i n wour LDA d a t a f i ' * 5 ' l e . ' / / ' ENTER THE FILENAME OF YOUR DATA F I L E (UP TO 8'. 6 ' CHARACTERS): ') 5110 FORMAT(/' Hope you e n j o y e d t h i s LDA p r o a r a a . Good l u c k 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 s e s s i o n . ' ) 6200 FORMAT(' CLASSIFICATION SCAN FOR F I L E •'.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. t X . S I l . 2 1 6 . IX. 511.216. IX. 511) Set ADC 0/P p o r t s 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 p r o a r a a p a r a a e t e r s f o r d e f a u l t v a l u e s . 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 ( f o r recorded l o c a t i o n s ) 9 ( f o r 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 c o n t r o l s speed o f scan w i t h 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. C o n t r o l o f speed and d i r e c t i o n with J o y s t i c k . C JVAL O/P from MSCAN p r o v i d e s 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 b e f o r e 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 a t 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  806 B30 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 o f 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 f o r low dose assay. CCCCC  C Open f i l e s 7 ( f o r c l a s s i f i c a t i o n s ) and 8 ( f o r 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 c a l c u l a t e c l a s s i f i c a t i o n f r e a u e n c i e s . 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  830  820  810 880  C C C 60  663  423  665  660  667 670 673  DO BSO 1-1,6 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 I DUM-1 DUM-1 WRITE(3,2100)ICLASS,NLOCIDUM IF<PARM(2).E0.O)G0TO 820 URITE(8 r 2100) ICLASS, NLOC, IDUM 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) CONTINUE IC-IC+1 IF<JDUM.NE.O.AND.IC.LT.6>00TO 802 ENDFILE 7 GOTO S Routine t o subisit f i l e BSLDAPRINT.OUT t o l i n e p r i n t REWIND B CALL 0UT50I27) CALL 0UT50(32> CALL 0UT50(32) NLINE-3 DO 663 I - l , S 3 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 PAGE<I) - FNPRN(I) CALL OUTSO(IO) CALL PLINE(PAGE,60) CALL OUTSO(IO) READ(8,4060.END-673) (LINE(I),1=1,80) IF(NLINE.LT.63)G0T0 670 NLINE-3 DO 660 1-1,4 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 CALL PLINE(PAGE.40> CALL OUTSOdO) NLINE-NLINEd CALL PLINE(LINE,80) GO TO 663 DO 680 I-NLINE,65  680  CALL 0UT30(10) REWIND 8 00 TO 3 C Set ADC 0/P p o r t s 24 and 23. 90 CALL 0UT(24.1S) CALL OUT(23.0> C Al1 done ' s i g n a l . CALL BEEP(5) U R T T F . =. . =. 1 m l  JT£>t» EMO  LDASUB  FOR  PAGE  300  LDASUB  01  CCCCCC AUTOMATIC SCAN. I/P * OF PULSES TO SUP..OX.DV. CCCCCC I n t e r r u p t s c a n anytime w i t h J o y s t i c k b u t t o n s . 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 p u l s e s t o motors when CDTN-l. 100 IF(NX.GE. lABS(DX) >CDTNX=0 IF(NY.GE.IABS(DY))CDTNV-0 I F ( C D T N X T C D T N Y . E Q . O I G O T O 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  C 120  C  C  PAGE 02  IF(XBIT.LT.0)XBIT-2  YBIT-0 IF(YDIR.LT.0)YBIT-1 IF(YDIR.GT.0)YBIT-2 L O O P . 0/P 0.1.2.4.fl.16.32.64 p u l s e s t o motors. (Depends on NX.NY). DO 170 1-1.64 CDTNX-0 CDTNY-0 CDTN SET. THIS CONTROLS MOTOR SPEED. NO MOTION UNLESS CDTNIF(((I*NX)/64)«44.EO.I»NX)CDTNX-1 IF<((I«NY)/64)«64.E0.I*NY)CDTNY=1 CHECK FOR STAGE OVERRUN•  C C  C C  1F(DX.OT.DXL)GOTO  190 170  HY-DYTYDIR*CDTNY  1B0  GOTO 120 RETURN END  140 130  500  130  IF(XDIR.E0.-11CDTNX-0 IF(DX.LT.DXH)GOTO 140 IF(XDIR,Ea.l)CDTNX-0 IF(DY.GT.DYL>GOTO 150 IF(YDIR.EO.-l)CDTNY-0 IF(DY.LT.DYH)G0TO 160 IF(YDIR.E0.1)CDTNY-0 SET OUPUT BYTE TO ADC ( U H I C H OUTPUTS B»XBIT»CBTNX+YBIT»CDTNY*4 B-15-B CALL 0UTC24.B) CALL 0UT(24.13) DX-DXtXDIR*CDTNX Clock t o c o n t r o l ranse o f speeds. DO 190 J - l . J E N D CONTINUE  130  CONTINUE  IEND=301-5PEED*30 DO 500 1=1.IEND 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  FOR  NY"0 XDIR"0 YDIR-0 IEND"301-30«SPEED INPUT FROM JOYSTICK. CALCULATE NX.NY FROM XJOY.YJOY. 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 I/P FROM JOYSTICK BUTTONS TO RETURN FROM SUBROUTINE. JVAL-INPC24) JUAL-JVAL-(JVAL/14 )* 14 IF( JVAL. LT.O) J V A L - J V A L M 6 IF(JVAL.NE.15)G0T0 1B0 IF(NXrNY.EO.0)GOTO 120 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  C 140  C  CCCCCC CCCCCC  TO MOTOR TRANSLATORS).  SIGNAL TI) OPERATOR FROM JOYSTICK SPEAKER• IEND DEFINES NUMBER OF BEEPS. BEEP{IEND)  SUBROUTINE  

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}]}"
                            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:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0085780/manifest

Comment

Related Items