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Cell Survival CurveDr. Vandana, King George’s Medical University, Lucknow
Cell Survival Curve   It describes relationship between radiation dose and    the fraction of cells that “survive” that d...
Cell DeathCell death can have different meanings:        loss of a specific function - differentiated cells (nerve,      ...
Relevant Dose       100 Gy           destroys cell function in non-proliferating systems (for            example: nerve,...
Survival   Conversely - “Survival” means retention of    reproductive integrity       the capacity for sustained prolife...
   Proof of reproductive integrity - the capability of a    single cell to grow into a large colony, visible to the    na...
Estimating Survival   In order to determine the surviving fraction, we    must know the plating efficiency   PE is the p...
Derivation of Survival Curves                             Always will have a control   Cells have been taken     batch to...
Surviving Fraction   Equal to the fraction of cells that plate    successfully and survive irradiation (without    losing...
72 colonies                         SF(2) =                            = 0.2                                    400 seeded...
   As Ionizations produced within cells by irradiation are    distributed randomly.   So consequently, cell death follow...
Quantization of cell killing   A dose of radiation that    introduces an average of one    lethal event per cell leaves  ...
   A straight line results when cell    survival (from a series of equal dose    fractions) is plotted on a logarithmic  ...
Survival Curve Features    Simple to describe qualitatively    Difficulty lies in explaining underlying biophysical     ...
Survival Curve Shape    general shapes of survival    curves for mammalian cells    exposed to radiation   Initial porti...
Mammalian Cell Survival Curve   Shoulder Region       Shows accumulation of SUB-        LETHAL DAMAGE.       The larger...
Two General Survival Models        Linear-quadratic model            “dual radiation action”            first component...
L-Q Model
Linear Quadratic Model                    2    S=    e-( D + D )    where:        S represents the fraction of cells su...
Linear Quadratic Model    Linear and quadratic contributions to cell     killing are equal when the dose is equal to the ...
and         Determination                      100                                                D           Survival    ...
Multi-target Model
Multi-target Model    Quantified in terms of:        measure of initial slope due to single-event killing,         D1   ...
D1 and D0 are  1. reciprocals of the initial and     final slopes  2. the doses required to reduce     the fraction of sur...
Multi-target Model   Shoulder-width measures:       the quasi-threshold dose (Dq)            the dose at which the extr...
Multi-Target Model                   n                                n or Dq represents the size                         ...
Linear –quadratic model      Multi-target model   Neither the L-Q not the M-T model has any established    biological bas...
Factors affecting cell survival curve 1. LET 2. Fractionation 3. Dose rate effect 4. Intrinsic radiosesitivity 5. Cell age...
LET    Low-LET radiations:        low dose region            shoulder region appears        high dose region         ...
   High-LET radiations:       survival curve is linear       surviving fraction is a pure exponential function of dose ...
Survival Curves and LET                     Increasing LET:                         increases the steepness             ...
Fractionation   If the dose is delivered as    equal fractions with sufficient      Dq    time ,repair of sub-lethal     ...
The Effective Survival Curve: Fractionation   If the dose is delivered as      Showing ~28 Gy    equal      fractions    ...
Dose-rate effectDose rate determines biological impact   reduction in dose rate causes reduced cell killing, due to    re...
Dose-Rate Effect in CHO CellsBroad shoulder to survivalcurve             Dose rate effect is more dramatic in CHO than in ...
Composite survival curves for 40 Human Cell Lines             Less variation      Low Dose Rate: Survival                 ...
Evidence for Dose Rate Effect in vivo: Crypt Cells                                        0.54 rad/min                    ...
Intrinsic radiosensitivityMammalian cells are significantly more radio-sensitivethan microorganisms:    Due to the differ...
Age response:Cell Cycle                                                         Late S—least sens.M>G2>G1>early S>late S f...
   Cells are most sensitive to radiation at or close to M   Cells are most resistant to radiation in late S   For prolo...
The Oxygen Effect   Oxygen modifies the biological effects of ionizing radiation   02 effect does not require that 02 be...
Cells are more sensitive toRadiation in the presence ofOxygen than in its absence                               High dose ...
Summary   A cell survival curve is the relationship between the fraction of    cells retaining their reproductive integri...
Summary   At low doses most cell killing results from “α-type” (single-hit, non-    repairable) injury, but that as the d...
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Cell survival curve
Cell survival curve
Cell survival curve
Cell survival curve
Cell survival curve
Cell survival curve
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Cell survival curve

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It describes relationship between radiation dose and the fraction of cells that “survive” that dose.
This is mainly used to assess biological effectiveness of radiation.
To understand it better, we need to know about a few basic things e.g.
Cell Death
Estimation of Survival / Plating Efficiency
Nature of Cell killing etc.

A cell survival curve is the relationship between the fraction of cells retaining their reproductive integrity and absorbed dose.

Conventionally, surviving fraction on a logarithmic scale is plotted on the Y-axis, the dose is on the X-axis . The shape of the survival curve is important.

The cell-survival curve for densely ionizing radiations (α-particles and low-energy neutrons) is a straight line on a log-linear plot, that is survival is an exponential function of dose.

The cell-survival curve for sparsely ionizing radiations (X-rays, gamma-rays has an initial slope, followed by a shoulder after which it tends to straighten again at higher doses.

Published in: Health & Medicine
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  • Well presented. My only criticism is you did not credit the original paper describing D0. Puck, T. T. and P. I. Marcus (1956). "Action of x-rays on mammalian cells." J Exp Med 103(5): 653-666.
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  • Excellent presentation, will be helpful for one and all.
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Transcript of "Cell survival curve"

  1. 1. Cell Survival CurveDr. Vandana, King George’s Medical University, Lucknow
  2. 2. Cell Survival Curve It describes relationship between radiation dose and the fraction of cells that “survive” that dose. This is mainly used to assess biological effectiveness of radiation. To understand it better, we need to know about a few basic things e.g.  Cell Death  Estimation of Survival / Plating Efficiency  Nature of Cell killing etc.
  3. 3. Cell DeathCell death can have different meanings:  loss of a specific function - differentiated cells (nerve, muscle, secretory cells)  loss of the ability to divide - proliferating cells such as stem cells in hematopoietic system or intestinal epithelium  loss of reproductive integrity - “reproductive death” 3
  4. 4. Relevant Dose 100 Gy  destroys cell function in non-proliferating systems (for example: nerve, muscle cells) 2 Gy  mean lethal dose for loss of proliferative capacity for proliferating cells 4
  5. 5. Survival Conversely - “Survival” means retention of reproductive integrity  the capacity for sustained proliferation in cells that proliferate5
  6. 6.  Proof of reproductive integrity - the capability of a single cell to grow into a large colony, visible to the naked eye A surviving cell that has retained its reproductive integrity and is able to proliferate indefinitely is said to be clonogenic6
  7. 7. Estimating Survival In order to determine the surviving fraction, we must know the plating efficiency PE is the percentage of cells (in control batch) that grow into colonies  in other words, those cells that survive the plating process7
  8. 8. Derivation of Survival Curves Always will have a control Cells have been taken batch to determine PE. from stock culture and placed in seed dishes Then irradiated (0 Gy to 6 Gy)and allowed to grow into colonies for 1-2 weeks Colonies have been counted for survival data 8
  9. 9. Surviving Fraction Equal to the fraction of cells that plate successfully and survive irradiation (without losing their reproductive integrity) to grow into colonies Colonies counted Surviving fraction cells seeded PE/100 9
  10. 10. 72 colonies SF(2) = = 0.2 400 seeded x 0.9 plated 1 0.1SurvivingFraction 0.01 0.001 2 4 6 Dose (Gy)10
  11. 11.  As Ionizations produced within cells by irradiation are distributed randomly. So consequently, cell death follows random probability statistics, the probability of survival decreasing geometrically with dose.
  12. 12. Quantization of cell killing A dose of radiation that introduces an average of one lethal event per cell leaves 37% still viable is called D0 dose. Cell killing follows exponential relationship. A dose which reduces cell survival to 50% will, if repeated, reduce survival to 25%, and similarly to 12.5% from a third exposure. This means Surviving fraction never becomes zero.
  13. 13.  A straight line results when cell survival (from a series of equal dose fractions) is plotted on a logarithmic scale as a function of dose on linear scale. The slope of such a semi-logarithmic dose curve could be described by the D0, the dose to reduce survival to 37%, D50, the dose to reduce survival to 50%, the D10, the dose to reduce survival to 10%. D0 usually lies between 1 and 2 Gy D10= 2.3 x D0
  14. 14. Survival Curve Features Simple to describe qualitatively Difficulty lies in explaining underlying biophysical events Many models have been proposed Steepness of curve represent the radio- sensitiveness. 14
  15. 15. Survival Curve Shape general shapes of survival curves for mammalian cells exposed to radiation Initial portion has a shoulder and terminal portion become straight line. In low dose region ,some dose of radiation goes waste. Terminal portion follow exponential relationship means same dose increment result into equal reduction in surviving fraction.
  16. 16. Mammalian Cell Survival Curve Shoulder Region  Shows accumulation of SUB- LETHAL DAMAGE.  The larger the shoulder region, the more dose will initally be needed to kill the same proportion of cells. Beyond the shoulder region  The D0 dose, or the inverse of the slop of the curve, indicates the relative radiosensitivity. The smaller the D0 dose, the greater the radiosensitivity.
  17. 17. Two General Survival Models  Linear-quadratic model  “dual radiation action”  first component - cell killing is proportional to dose  second component - cell killing is proportional to dose squared  Multi-target model  based on probability of hitting the “target”  widely used for many years; still has merit17
  18. 18. L-Q Model
  19. 19. Linear Quadratic Model 2 S= e-( D + D ) where:  S represents the fraction of cells surviving  D represents dose  and are constants that characterize the slopes of the two l portions of the semi-log survival curve  biological endpoint is cell death 22
  20. 20. Linear Quadratic Model Linear and quadratic contributions to cell killing are equal when the dose is equal to the ratio of to  D = / or  D = D2  component is representative of damage caused by a single event (hit, double-strand break, “initiation / promotion” etc.)  component is representative of damage caused by multiple events (hit/hit, 2 strand breaks, initiation then promotion, etc.)23
  21. 21. and Determination 100 D Survival 10-1 D2 10-2 0 3 6 9 12 Dose, Gy24
  22. 22. Multi-target Model
  23. 23. Multi-target Model Quantified in terms of:  measure of initial slope due to single-event killing, D1  measure of final slope due to multiple-event killing, D0  width of the shoulder, Dq or n28
  24. 24. D1 and D0 are 1. reciprocals of the initial and final slopes 2. the doses required to reduce the fraction of surviving cells by 37% 3. the dose required to deliver, on average, one inactivating event per cell 4. D1,reduces survivivig fraction to 0.37 5. D0, from 0.1 to 0.037, or from 0.01 to 0.0037 ,and so on.
  25. 25. Multi-target Model Shoulder-width measures:  the quasi-threshold dose (Dq)  the dose at which the extrapolated line from the straight portion of the survival curve (final slope) crosses the axis at 100% survival  the extrapolation number (n)  This value is obtained by extrapolating the exponential portion of the curve to the vertical line.  “broad shoulder” results in larger value of n  “narrow shoulder” results in small value of n  n = exp[Dq / D0] 30
  26. 26. Multi-Target Model n n or Dq represents the size or width of the shoulder Dq 100 10-1 Initial slope measure, D1, Survival due to single-event killing 10-2 Final slope measure, D0, 10-3 due to multiple-event killing 10-4 0 3 6 9 12 Dose, Gy31
  27. 27. Linear –quadratic model Multi-target model Neither the L-Q not the M-T model has any established biological basis. At high doses the LQ model predicts a survival curve that bends continuosly, whereas the M-T model become linear. At low doses the LQ model describes a curve that bends more than a M-T curve.
  28. 28. Factors affecting cell survival curve 1. LET 2. Fractionation 3. Dose rate effect 4. Intrinsic radiosesitivity 5. Cell age 6. Oxygen presence
  29. 29. LET Low-LET radiations:  low dose region  shoulder region appears  high dose region  survival curve becomes linear and surviving fraction to an exponential function of dose  surviving fraction is a dual exponential S = e-( D+ D2)34
  30. 30.  High-LET radiations:  survival curve is linear  surviving fraction is a pure exponential function of dose S = e-( D) 35
  31. 31. Survival Curves and LET  Increasing LET:  increases the steepness of the survival curve  results in a more linear curve  shoulder disappears due to increase of killing by single-events36
  32. 32. Fractionation If the dose is delivered as equal fractions with sufficient Dq time ,repair of sub-lethal n = exp[Dq / D0] damage ocurs 104 103 Elkind‟ s Recovery takes place 102 between radiation exposure , Dq 101 cell act as fresh target. 100 10-1 D0 Elkind & Sutton showed that when two exposure were given 10-2 5 10 15 25 20 few hours apart ,the shoulder Dose (Gy) reappeared.
  33. 33. The Effective Survival Curve: Fractionation If the dose is delivered as Showing ~28 Gy equal fractions with in 14 fractions. sufficient time between for repair of the sub-lethal (non- killing) damage, the shoulder of the survival curve is repeated many times. The effective survival curve becomes a composite of all the shoulder repetitions. Dose required to produce the same reduction in surviving fraction increases. D0 is 3 Gy 38
  34. 34. Dose-rate effectDose rate determines biological impact reduction in dose rate causes reduced cell killing, due to repair of SLD reduction in dose rate generally reduces survival-curve slope (D0 increases) inverse dose-rate effect occurs in some cell lines at „optimal‟ dose rate due to accumulation of cells in G2
  35. 35. Dose-Rate Effect in CHO CellsBroad shoulder to survivalcurve Dose rate effect is more dramatic in CHO than in HeLa Cells
  36. 36. Composite survival curves for 40 Human Cell Lines Less variation Low Dose Rate: Survival Curves show greater variation, Greater range in repair times
  37. 37. Evidence for Dose Rate Effect in vivo: Crypt Cells 0.54 rad/min 0.92 Crypt cells: rapid dividing Dramatic dose rate effect Exposure time is longer than 4.5 Cell cycle (repopulation) 36 274
  38. 38. Intrinsic radiosensitivityMammalian cells are significantly more radio-sensitivethan microorganisms:  Due to the differences in DNA content  represents bigger target for radiation damage  Sterilizing radiation dose for bacteria is 20,000 Gy
  39. 39. Age response:Cell Cycle Late S—least sens.M>G2>G1>early S>late S for sensitivityDifference caused by cell cycle are similar to difference caused by Oxygen effect
  40. 40.  Cells are most sensitive to radiation at or close to M Cells are most resistant to radiation in late S For prolonged G1  a resistant period is evident early G1 followed be a sensitive period in late G1 Cells are usually sensitive to radiation in G2 (almost as sensitive as in M)
  41. 41. The Oxygen Effect Oxygen modifies the biological effects of ionizing radiation 02 effect does not require that 02 be present during radiation – just added within 5 msec after generation of free radical OER – oxygen enhancement ratio: ratio of hypoxic: aerated doses needed to achieve the same biological effect X-Rays/γ-Rays  at high doses is 2.5-3.5 OER is ~2.5 at lower doses OER is absent for high LET radiations like alpha-particles and is intermediate for fast neutron. OER is lower for types of radiation predisposed to killing cells by single-hit mechanisms
  42. 42. Cells are more sensitive toRadiation in the presence ofOxygen than in its absence High dose region of survival curve Low dose region of survival curve
  43. 43. Summary A cell survival curve is the relationship between the fraction of cells retaining their reproductive integrity and absorbed dose. Conventionally, surviving fraction on a logarithmic scale is plotted on the Y-axis, the dose is on the X-axis . The shape of the survival curve is important. The cell-survival curve for densely ionizing radiations (α- particles and low-energy neutrons) is a straight line on a log- linear plot, that is survival is an exponential function of dose. The cell-survival curve for sparsely ionizing radiations (X- rays, gamma-rays has an initial slope, followed by a shoulder after which it tends to straighten again at higher doses.
  44. 44. Summary At low doses most cell killing results from “α-type” (single-hit, non- repairable) injury, but that as the dose increases, the“β –type” (multi-hit, repairable) injury becomes predominant, increasing as the square of the dose. Survival data are fitted by many models. Some of them are: multitarget hypothesis, linear-quadratic hypothesis. The survival curve for a multifraction regimen is also an exponential function of dose. The D10, the dose resulting in one decade of cell killing, is related to the Do by the expression D10 = 2.3 x Do Cell survival also depends on the dose, dose rate and the cell type
  45. 45. Thank You
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