1975, British Journal of Radiology, 48, 65
JANUARY 1975
Correspondence (The Editors do not hold themselves responsible for opinions expressed by correspondents) THE EDITOR—SIR, THE "SELF-REPORTING RADIOGRAPH"
A new pictorial dimension to the radiologist's report. "One picture may be worth a thousand words" but review of radiographs by clinicians without the expert interpretation of a radiologist may often be misleading. The following method of an instantaneous pictorial supplement to the radiologist's report was, therefore, adopted. The report is dictated in the usual manner. However, whenever pertinent pathological findings are noted, the radiologist selects one or several films on which they are well depicted and denotes them by means of salient notations made with erasable wax pencil directly on the film. A well-placed arrow and a few key words often suffice to concisely convey the most important findings (Fig. 1A). The selected film or films are then clipped outside the X-ray envelope with instructions to be duplicated. An X-ray copier (Delcomat 100 mm X-ray Copier) which reduces standard-sized films to 100x100 mm direct positive copy film may be utilized (Fig. 1B) . These 100 X 100 mm direct film copies are easily made, in broad daylight, and can be immediately processed in most late model 90second automatic X-ray processors (Fig. lc) or in small film processors. The 100x100 mm copy is then secured to a blank report form or to the actual dictated report sheet, if already available, and then sent directly to the patient's chart (Fig. ID) where it becomes part of the permanent record. The 100 X 100 mm copy can be easily viewed by the medical staff by simply moving the transparency away from the report sheet and holding it against a light source. An alternate method is to use another X-ray copier (LogEtronic Inc. Systems and X-ray Copier) capable of producing 105 X 140 mm intermediate "black bone" negatives. These can then be processed into positive transparencies or prints which are then sent to the charts. The film mini-copies are relatively inexpensive, and despite their small size, the mini-copies have good resolution and sharp detail. They can furthermore be comfortably viewed with the naked eye and the radiologist's notations
FIG. 1A-D.
65
clearly discerned without the aid of magnifying lenses or projectors. The annotated mini-copies may also be used to good advantage in lieu of the usual film duplicates, whenever radiographs are needed for such diverse purposes as museum files, loans to medical staff or mailings to other institutions with patient transfers or consultations. In the latter instance, they are both easier and cheaper to send by mail. Another potential value of the miniature "Self-Reporting Radiograph" may be its ability to do double duty as a substitute for microfilming of radiographs at a significantly reduced cost. One miniature copy or print with the radiologist's annotations could be placed in the patient's chart and the other in a microfilm file or "black bone" masterfile in the Radiology Department. The chief drawbacks of currently used microfilming methods include the necessity of additional optical aids for viewing with attendant loss of original information, separate retrieval of reports and the superfluous duplication of all radiographs. These drawbacks are obviated with the "SelfReporting Radiographs" which serve an immediate useful function while the case is still active. Yours, etc., RUBEM POCHACZEVSKY.
Department of Radiology, City Hospital Center at Elmhurst, 79-01 Broadway, Elmhurst, N.Y.I 1373.
THE EDITOR—SIR, APPLICATION OF FLOW MICROFLUOROMETRY TO PROBLEMS IN RADIOTHERAPY
There is currently increasing interest in the use of flow microfluorometry (FMF) instrumentation for rapid and quantitative measurement of DNA content, protein content, etc., of individual cells (Mullaney et al., 1974). Fluorescently stained cells pass singly through a focused argon-ion laser beam. The fluorescent light is collected at right angles to the laser beam, and the intensity is measured by a photomultiplier tube coupled through amplifiers to a multichannel analyser. The fluorescent intensity (corresponding to channel number) is proportional to the DNA content of each cell. This type of instrumentation has been used to measure the cell cycle distribution changes in a rat rhabdomyosarcoma (in vivo and in vitro) after radiation exposure (Kal, 1973a, b). We have reported previously the changes of age distribution of KHT sarcoma tumour cells with time after radiation treatment (Raju et al., 1974). The present preliminary study describes changes in normal and tumour cell populations of an 85-year-old American Indian patient with carcinoma of the cervix during the course of radiotherapy. Preliminary studies (Horan and Romero, 1974) using FMF instrumentation indicated that at least some Burkett lymphoma and rhabdomyosarcoma have a DNA content which is nearly the same as normal cells. However, a significant fraction of tumours studied, such as squamous cell carcinoma of the cervix (Atkins, 1964), lung carcinomas, and breast carcinomas generally have elevated DNA content. Since normal cells and tumour cells are present simultaneously in specimens, DNA content as measured by FMF instrumentation is useful to detect changes in cell populations resulting from radiotherapy if tumours have elevated DNA content. In other studies, it was shown (Horan et al., 1974) that it is possible to detect 1 per cent tumour cells in
VOL.
48, No. 565 Correspondence
a sample containing 99 per cent normal cells when the abnormal cell population has more DNA per cell than the normal cells. Previous experience has shown (Romero and Horan, 1974) that exfoliated cervical cells can be disaggregated using the enzyme collagenase. Also, cells from squamous cell carcinoma have more DNA per cell as measured by FMF. With this information and the previous results on animal tumours, we began to study the effects of radiotherapy on squamous cell carcinoma of the cervix. In all samples the DNA was stained using the acriflavine-Feulgen technique (Kraemer, Petersen and Van Dilla, 1971). These were analysed with the FMF instrumentation. Figure 1A shows the DNA distribution of exfoliated cells from a normal patient, and Fig. 1B—IF are from a patient with squamous cell carcinoma during stages of radiotherapy treatment. The x axis in these figures is the channel number proportional to DNA content, and the y axis is the cell
number. Two peaks are observed in Fig. 1A, one small peak corresponding to channel No. 11 and another large peak corresponding to channel No. 19. By separating out the cells in these peaks using the cell separator (Steinkamp et ah. 1973), it has been confirmed that the cells in the first peak were leucocytes and that cells in the large peak were normal squamous cells. Strictly speaking, both leucocytes and normal cells should have the same DNA content. Leucocytes seem to stain less intensely than normal exfoliated cells; hence, the peak due to leucocytes occurred at a lower channel number than squamous cells. This is a staining artifact (Deitch, Wagner and Richart, 1968). Figure 1B shows results after two daily treatments of 300 rads each. The peak in channel No. 28 corresponds to the G\ phase of the tumour cells, and the DNA content of tumour cells in this phase was nearly 1-5 times that found for normal squamous cells. There was also another peak in channel No. 58 (nearly twice the amount of Gi), and this
(8)Carcinoma of the cervix 600 rads 9/14/73
10
10 (C) Same patient os in B 2100 rads 10/1/73
(A) Normal
0.3 Abn (D) Same patient as in B 2700 rads 10/9/73
(E)Same patient as in B 3450 rods 10/16/73
0.2
Q2 (F) Same patient as in B 4200 rads 10/19/73
O.I Abn
0.1
'0
20
40
60
80
Channel Number (DNA content) FIG. 1. DNA profiles of squamous cells after acriflavine-Feulgen staining, (A) A normal patient. The specimen contained superficial squamous cells and leucocytes, (B-F) DNA profiles of specimens taken from an 85-year-old Indian woman with carcinoma of the cervix. Dates of specimen collection and total accumulated dose the patient received are given in each panel. The smooth curves are not computer-fitted but drawn by the illustrator.
66
1975, British Journal of Radiology, 48, 67-68
JANUARY 1975
Correspondence corresponds to tumour cells in the G2 + M phase. Cells in between these two peaks were in .S phase. A significant fraction of cells were cycling. It should be noted that very few normal cells were present in this sample. Cytological examination of this sample indicated cells with malignant characteristics and a small fraction of cells showing irradiation effects. After 2,100 rads of treatment (Fig. lc), a large leucocyte peak was seen. At this point a uterine infection was observed, giving rise to a large number of leucocytes in the vaginal area. It should be noted also that a large amount of fluorescent material was seen in channels Nos. 1—5. This was fluorescent debris which may be a result of radiationinduced necrosis. Furthermore, it should be noted that no distinct peaks were evident corresponding to the DNA content of normal or abnormal cells. However, comparison of Fig. lc with Fig. 1A suggests that a large number of tumour cells are present (Fig. lc, channel No. 50). The cells in this region may be the giant cells often seen in cervical Papanicolaou smears from irradiated patients. Cytological examination revealed squamous carcinoma cells, parabasal cells, leucocytes, intermediate cells and debris. After 2,700 rads of radiotherapy (Fig. ID), debris and leucocytes are present. However, at this point there is an indication of a peak due to abnormal cells. With continuing treatment (Fig. 1E), it was evident that the abnormal peak was diminishing in intensity. However, no normal cell peak was present. Cytological observations of this sample were similar to that after 2,100 rads of treatment. After 4,200 rads of radiotherapy (Fig. IF), a distinct peak of normal cells was present, and no signs of the uterine infection were seen at this time. It should be noted that no leucocyte peak was present. While no discrete peak of abnormal cells was present in this specimen, detailed analysis indicated that nearly 8 per cent of the cells in this specimen were abnormal. Cytological examination of this sample also indicated a large population of white cells and a small percentage of keratinizing malignant cells. At the termination of treatment (11/27/73, 9,720 rads), no abnormal cells were detected by FMF or classical Papanicolaou examination. These results clearly indicate that in tumours with elevated DNA content FMF analysis could be very useful to monitor changes quantitatively in the distribution of tumour and normal cells during the course of radiotherapy, although the changes in exfoliated cell populations may not strictly represent changes in the tumour. Nearly optimum fractionation schedules and integrated doses in radiotherapy using conventional radiations were derived empirically from 50 years of experience. By monitoring the changes quantitatively with FMF along with cytopathological observations during the course of radiotherapy with conventional radiations, one could probably use these data to help decide the course of radiotherapy treatment with unconventional radiations such as fast neutrons, negative pions and heavy ions. Yours, etc.
REFERENCES ATKINS, N. B., 1964. The DNA content of malignant cells in cervical smears. Ada Cytologica, 8, 68-72. DEITCH, A. D., WAGNER, D., and RICHART, R. M., 1968.
Conditions influencing the intensity of the Feulgen reaction. Journal of Histochemistry Cytochemistry, 16, 371—379. HORAN, P. K., ROMERO, A., STEINKAMP, J., and PETERSEN,
D., 1974. Detection of heteroploid tumor cells. Journal of the National Cancer Institute, 52, 843-848. HORAN, P. K., and ROMERO, A., 1974. DNA content of
M. R. RAJU, P. K. HORAN, A. ROMERO. J. C. MARTIN.
Biophysics and Instrumentation Group, Los Alamos Scientific Laboratory, University of California, Los Alamos, New Mexico 87544. C. J. STERNHAGEN.
Cancer Research and Treatment Center, University of New Mexico, School of Medicine, Albuquerque, New Mexico 87106. ACKNOWLEDGMENT
This work was performed under AEC/NCI agreement No. 2-YO1-CB-10055-03 (formerly NCI-GLC-(71)-55). 67
tumor cells (in preparation). KAL, H. B., 1973a. Distributions of cell volume and DNA content of rhabdomyosarcoma cells growing in vitro and in vivo after irradiation. European Journal of Cancer, 9, 77-79. 1973b. Proliferation behaviour of P and Q cells in a rat rhabdomyosarcoma after irradiation as determined by DNA measurements. European Journal of Cancer, 9, 753-756. KRAEMER, P. M., PETERSEN, D. F., and VAN DILLA, M. A.
1971. On DNA constancy in heteroploidy and the stemline theory of tumours. Science, 174, IXA—lXl. MULLANEY, P. F., STEINKAMP, J. A., CRISSMAN, H. A., CRAM,
L. S., and HOLM, D. M., 1974. Laser flow microphotometers for rapid analysis and sorting of individual mammalian cells. In Laser Applications in Medicine and Biology, ed. M. L. Wolbarsht (Plenum Press, New York), in press. RAJU, M. R., TRUJILLO, T. T., MULLANEY, P. F., ROMERO, A., STEINKAMP, J. A., and WALTERS, R. A., 1974. The
distribution in the cell cycle of normal cells and of irradiated tumour cells in mice. British Journal of Radiology, 47, 405-110. ROMERO, A., and HORAN, P. K., 1974. Disaggregation of
cervical squamous cells (in preparation). STEINKAMP, J. A., FULWYLER, M. J., COULTER, J. R., HIEBERT, R. D., HORNEY, J. L., and MULLANEY, P. F.,
1973. A new parameter separator for microscopic particles and biological cells. Review of Scientific Instruments, 44, 1301-1310.
THE EDITOR—SIR, THE NSD FORMULA AND THE OXFORD PIG SKIN EXPERIMENTS
In a recent paper (Berry etal., 19741) pigskin experiments led to the conclusion that the NSD formula failed to correctly represent iso-effect doses in the range of 6—30 fractions. Unfortunately, the authors did not discuss the statistical significance of their results. In fact, my own analysis of their data shows that precisely the opposite conclusion could be drawn, namely, the NSD formula does represent iso-effect doses calculated from their results zcithin experimental error. Rather than disputing all of the conclusions they have made from their data, for the sake of brevity I will consider the most important, specifically, that the slope of the isoeffect line between six and 30 fractions is 0-46, and that errors of greater than 30 per cent could result if the formula were used in clinical practice. These conclusions are based entirely on the data they quote for 6f./18d and 30f./39d, and this is reproduced in my Table I. For the construction-of their iso-effect curve (Fig. 2) they chose a fractional reduction of 078 as the standard biological effect. With the 6f./18d regime this is produced by a dose of 3,790 rads, on average. With the 30f./39d scheme, their choice of approximately 8,000 rads as the iso-effect dose appears arbitrary, since 8,000 rads gave a 0-73 reduction in field size. Furthermore, this represented only one result from one animal and the results from the other five animals in this group appear to be ignored. In order to use the 30f./39d data from all six animals, I analysed their results by determining the regression line correlating dose with the observed fractional reduction of the field.
JANUARY 1975
Correspondence (The Editors do not hold themselves responsible for opinions expressed by correspondents) THE EDITOR—SIR, THE "SELF-REPORTING RADIOGRAPH"
A new pictorial dimension to the radiologist's report. "One picture may be worth a thousand words" but review of radiographs by clinicians without the expert interpretation of a radiologist may often be misleading. The following method of an instantaneous pictorial supplement to the radiologist's report was, therefore, adopted. The report is dictated in the usual manner. However, whenever pertinent pathological findings are noted, the radiologist selects one or several films on which they are well depicted and denotes them by means of salient notations made with erasable wax pencil directly on the film. A well-placed arrow and a few key words often suffice to concisely convey the most important findings (Fig. 1A). The selected film or films are then clipped outside the X-ray envelope with instructions to be duplicated. An X-ray copier (Delcomat 100 mm X-ray Copier) which reduces standard-sized films to 100x100 mm direct positive copy film may be utilized (Fig. 1B) . These 100 X 100 mm direct film copies are easily made, in broad daylight, and can be immediately processed in most late model 90second automatic X-ray processors (Fig. lc) or in small film processors. The 100x100 mm copy is then secured to a blank report form or to the actual dictated report sheet, if already available, and then sent directly to the patient's chart (Fig. ID) where it becomes part of the permanent record. The 100 X 100 mm copy can be easily viewed by the medical staff by simply moving the transparency away from the report sheet and holding it against a light source. An alternate method is to use another X-ray copier (LogEtronic Inc. Systems and X-ray Copier) capable of producing 105 X 140 mm intermediate "black bone" negatives. These can then be processed into positive transparencies or prints which are then sent to the charts. The film mini-copies are relatively inexpensive, and despite their small size, the mini-copies have good resolution and sharp detail. They can furthermore be comfortably viewed with the naked eye and the radiologist's notations
FIG. 1A-D.
65
clearly discerned without the aid of magnifying lenses or projectors. The annotated mini-copies may also be used to good advantage in lieu of the usual film duplicates, whenever radiographs are needed for such diverse purposes as museum files, loans to medical staff or mailings to other institutions with patient transfers or consultations. In the latter instance, they are both easier and cheaper to send by mail. Another potential value of the miniature "Self-Reporting Radiograph" may be its ability to do double duty as a substitute for microfilming of radiographs at a significantly reduced cost. One miniature copy or print with the radiologist's annotations could be placed in the patient's chart and the other in a microfilm file or "black bone" masterfile in the Radiology Department. The chief drawbacks of currently used microfilming methods include the necessity of additional optical aids for viewing with attendant loss of original information, separate retrieval of reports and the superfluous duplication of all radiographs. These drawbacks are obviated with the "SelfReporting Radiographs" which serve an immediate useful function while the case is still active. Yours, etc., RUBEM POCHACZEVSKY.
Department of Radiology, City Hospital Center at Elmhurst, 79-01 Broadway, Elmhurst, N.Y.I 1373.
THE EDITOR—SIR, APPLICATION OF FLOW MICROFLUOROMETRY TO PROBLEMS IN RADIOTHERAPY
There is currently increasing interest in the use of flow microfluorometry (FMF) instrumentation for rapid and quantitative measurement of DNA content, protein content, etc., of individual cells (Mullaney et al., 1974). Fluorescently stained cells pass singly through a focused argon-ion laser beam. The fluorescent light is collected at right angles to the laser beam, and the intensity is measured by a photomultiplier tube coupled through amplifiers to a multichannel analyser. The fluorescent intensity (corresponding to channel number) is proportional to the DNA content of each cell. This type of instrumentation has been used to measure the cell cycle distribution changes in a rat rhabdomyosarcoma (in vivo and in vitro) after radiation exposure (Kal, 1973a, b). We have reported previously the changes of age distribution of KHT sarcoma tumour cells with time after radiation treatment (Raju et al., 1974). The present preliminary study describes changes in normal and tumour cell populations of an 85-year-old American Indian patient with carcinoma of the cervix during the course of radiotherapy. Preliminary studies (Horan and Romero, 1974) using FMF instrumentation indicated that at least some Burkett lymphoma and rhabdomyosarcoma have a DNA content which is nearly the same as normal cells. However, a significant fraction of tumours studied, such as squamous cell carcinoma of the cervix (Atkins, 1964), lung carcinomas, and breast carcinomas generally have elevated DNA content. Since normal cells and tumour cells are present simultaneously in specimens, DNA content as measured by FMF instrumentation is useful to detect changes in cell populations resulting from radiotherapy if tumours have elevated DNA content. In other studies, it was shown (Horan et al., 1974) that it is possible to detect 1 per cent tumour cells in
VOL.
48, No. 565 Correspondence
a sample containing 99 per cent normal cells when the abnormal cell population has more DNA per cell than the normal cells. Previous experience has shown (Romero and Horan, 1974) that exfoliated cervical cells can be disaggregated using the enzyme collagenase. Also, cells from squamous cell carcinoma have more DNA per cell as measured by FMF. With this information and the previous results on animal tumours, we began to study the effects of radiotherapy on squamous cell carcinoma of the cervix. In all samples the DNA was stained using the acriflavine-Feulgen technique (Kraemer, Petersen and Van Dilla, 1971). These were analysed with the FMF instrumentation. Figure 1A shows the DNA distribution of exfoliated cells from a normal patient, and Fig. 1B—IF are from a patient with squamous cell carcinoma during stages of radiotherapy treatment. The x axis in these figures is the channel number proportional to DNA content, and the y axis is the cell
number. Two peaks are observed in Fig. 1A, one small peak corresponding to channel No. 11 and another large peak corresponding to channel No. 19. By separating out the cells in these peaks using the cell separator (Steinkamp et ah. 1973), it has been confirmed that the cells in the first peak were leucocytes and that cells in the large peak were normal squamous cells. Strictly speaking, both leucocytes and normal cells should have the same DNA content. Leucocytes seem to stain less intensely than normal exfoliated cells; hence, the peak due to leucocytes occurred at a lower channel number than squamous cells. This is a staining artifact (Deitch, Wagner and Richart, 1968). Figure 1B shows results after two daily treatments of 300 rads each. The peak in channel No. 28 corresponds to the G\ phase of the tumour cells, and the DNA content of tumour cells in this phase was nearly 1-5 times that found for normal squamous cells. There was also another peak in channel No. 58 (nearly twice the amount of Gi), and this
(8)Carcinoma of the cervix 600 rads 9/14/73
10
10 (C) Same patient os in B 2100 rads 10/1/73
(A) Normal
0.3 Abn (D) Same patient as in B 2700 rads 10/9/73
(E)Same patient as in B 3450 rods 10/16/73
0.2
Q2 (F) Same patient as in B 4200 rads 10/19/73
O.I Abn
0.1
'0
20
40
60
80
Channel Number (DNA content) FIG. 1. DNA profiles of squamous cells after acriflavine-Feulgen staining, (A) A normal patient. The specimen contained superficial squamous cells and leucocytes, (B-F) DNA profiles of specimens taken from an 85-year-old Indian woman with carcinoma of the cervix. Dates of specimen collection and total accumulated dose the patient received are given in each panel. The smooth curves are not computer-fitted but drawn by the illustrator.
66
1975, British Journal of Radiology, 48, 67-68
JANUARY 1975
Correspondence corresponds to tumour cells in the G2 + M phase. Cells in between these two peaks were in .S phase. A significant fraction of cells were cycling. It should be noted that very few normal cells were present in this sample. Cytological examination of this sample indicated cells with malignant characteristics and a small fraction of cells showing irradiation effects. After 2,100 rads of treatment (Fig. lc), a large leucocyte peak was seen. At this point a uterine infection was observed, giving rise to a large number of leucocytes in the vaginal area. It should be noted also that a large amount of fluorescent material was seen in channels Nos. 1—5. This was fluorescent debris which may be a result of radiationinduced necrosis. Furthermore, it should be noted that no distinct peaks were evident corresponding to the DNA content of normal or abnormal cells. However, comparison of Fig. lc with Fig. 1A suggests that a large number of tumour cells are present (Fig. lc, channel No. 50). The cells in this region may be the giant cells often seen in cervical Papanicolaou smears from irradiated patients. Cytological examination revealed squamous carcinoma cells, parabasal cells, leucocytes, intermediate cells and debris. After 2,700 rads of radiotherapy (Fig. ID), debris and leucocytes are present. However, at this point there is an indication of a peak due to abnormal cells. With continuing treatment (Fig. 1E), it was evident that the abnormal peak was diminishing in intensity. However, no normal cell peak was present. Cytological observations of this sample were similar to that after 2,100 rads of treatment. After 4,200 rads of radiotherapy (Fig. IF), a distinct peak of normal cells was present, and no signs of the uterine infection were seen at this time. It should be noted that no leucocyte peak was present. While no discrete peak of abnormal cells was present in this specimen, detailed analysis indicated that nearly 8 per cent of the cells in this specimen were abnormal. Cytological examination of this sample also indicated a large population of white cells and a small percentage of keratinizing malignant cells. At the termination of treatment (11/27/73, 9,720 rads), no abnormal cells were detected by FMF or classical Papanicolaou examination. These results clearly indicate that in tumours with elevated DNA content FMF analysis could be very useful to monitor changes quantitatively in the distribution of tumour and normal cells during the course of radiotherapy, although the changes in exfoliated cell populations may not strictly represent changes in the tumour. Nearly optimum fractionation schedules and integrated doses in radiotherapy using conventional radiations were derived empirically from 50 years of experience. By monitoring the changes quantitatively with FMF along with cytopathological observations during the course of radiotherapy with conventional radiations, one could probably use these data to help decide the course of radiotherapy treatment with unconventional radiations such as fast neutrons, negative pions and heavy ions. Yours, etc.
REFERENCES ATKINS, N. B., 1964. The DNA content of malignant cells in cervical smears. Ada Cytologica, 8, 68-72. DEITCH, A. D., WAGNER, D., and RICHART, R. M., 1968.
Conditions influencing the intensity of the Feulgen reaction. Journal of Histochemistry Cytochemistry, 16, 371—379. HORAN, P. K., ROMERO, A., STEINKAMP, J., and PETERSEN,
D., 1974. Detection of heteroploid tumor cells. Journal of the National Cancer Institute, 52, 843-848. HORAN, P. K., and ROMERO, A., 1974. DNA content of
M. R. RAJU, P. K. HORAN, A. ROMERO. J. C. MARTIN.
Biophysics and Instrumentation Group, Los Alamos Scientific Laboratory, University of California, Los Alamos, New Mexico 87544. C. J. STERNHAGEN.
Cancer Research and Treatment Center, University of New Mexico, School of Medicine, Albuquerque, New Mexico 87106. ACKNOWLEDGMENT
This work was performed under AEC/NCI agreement No. 2-YO1-CB-10055-03 (formerly NCI-GLC-(71)-55). 67
tumor cells (in preparation). KAL, H. B., 1973a. Distributions of cell volume and DNA content of rhabdomyosarcoma cells growing in vitro and in vivo after irradiation. European Journal of Cancer, 9, 77-79. 1973b. Proliferation behaviour of P and Q cells in a rat rhabdomyosarcoma after irradiation as determined by DNA measurements. European Journal of Cancer, 9, 753-756. KRAEMER, P. M., PETERSEN, D. F., and VAN DILLA, M. A.
1971. On DNA constancy in heteroploidy and the stemline theory of tumours. Science, 174, IXA—lXl. MULLANEY, P. F., STEINKAMP, J. A., CRISSMAN, H. A., CRAM,
L. S., and HOLM, D. M., 1974. Laser flow microphotometers for rapid analysis and sorting of individual mammalian cells. In Laser Applications in Medicine and Biology, ed. M. L. Wolbarsht (Plenum Press, New York), in press. RAJU, M. R., TRUJILLO, T. T., MULLANEY, P. F., ROMERO, A., STEINKAMP, J. A., and WALTERS, R. A., 1974. The
distribution in the cell cycle of normal cells and of irradiated tumour cells in mice. British Journal of Radiology, 47, 405-110. ROMERO, A., and HORAN, P. K., 1974. Disaggregation of
cervical squamous cells (in preparation). STEINKAMP, J. A., FULWYLER, M. J., COULTER, J. R., HIEBERT, R. D., HORNEY, J. L., and MULLANEY, P. F.,
1973. A new parameter separator for microscopic particles and biological cells. Review of Scientific Instruments, 44, 1301-1310.
THE EDITOR—SIR, THE NSD FORMULA AND THE OXFORD PIG SKIN EXPERIMENTS
In a recent paper (Berry etal., 19741) pigskin experiments led to the conclusion that the NSD formula failed to correctly represent iso-effect doses in the range of 6—30 fractions. Unfortunately, the authors did not discuss the statistical significance of their results. In fact, my own analysis of their data shows that precisely the opposite conclusion could be drawn, namely, the NSD formula does represent iso-effect doses calculated from their results zcithin experimental error. Rather than disputing all of the conclusions they have made from their data, for the sake of brevity I will consider the most important, specifically, that the slope of the isoeffect line between six and 30 fractions is 0-46, and that errors of greater than 30 per cent could result if the formula were used in clinical practice. These conclusions are based entirely on the data they quote for 6f./18d and 30f./39d, and this is reproduced in my Table I. For the construction-of their iso-effect curve (Fig. 2) they chose a fractional reduction of 078 as the standard biological effect. With the 6f./18d regime this is produced by a dose of 3,790 rads, on average. With the 30f./39d scheme, their choice of approximately 8,000 rads as the iso-effect dose appears arbitrary, since 8,000 rads gave a 0-73 reduction in field size. Furthermore, this represented only one result from one animal and the results from the other five animals in this group appear to be ignored. In order to use the 30f./39d data from all six animals, I analysed their results by determining the regression line correlating dose with the observed fractional reduction of the field.
