STUDIES I N MOUSE LEUKEMIA

LYMPHATIC LEUKEMIA X. METABOLIC DIFFERENCES BETWEEN TRANSMISSION LINES OF MOUSE

.IOSEPH VICTOR AND MARGARET R. WINTERSTEINER

(From the Department of Pathology, College of Pliyaicians and Surgeom, Columbia Univer- sity, New Pork, New Pork, and the Department of Genetics, Carnegie

Institution of Washiitgton, Cold Spiing Harbor)

Metabolic studies in leukemia have been frequent since the first clinical applications of calorimetry. One of the first patients studied in a calorimeter (Pettenkofer and Voit, 1869, 21) had leukemia. The increased basal metabolic rate in both myeloid and lymphatic leukemia is well established and seems to be correlated with the severity of the disease. There appears to be some disagreement as to the mechanism concerned in this altered metabolism. A large group of investigators attribute the increased metabolism to changes in the abnormal cells concerned (6, 8, 10, 25, 26). Others ascribe it to effects secondary to disturbed endocrine activity (9).

There have been three methods of approach to this problem, the first, in v i m ; the second and third, in vitro. First, the metabolism of the entire animal has been investigated, yielding the resultant of a mixed tissue metabolism (11, 15, 16,19). The second method has been concerned with the metabolism of the leukemic cells in the blood o r tissues infiltrated by these cells (1, 2,3, 6,8,10,12,18, 20, 25, 26, 27, 34). In a few instances, attempts have been made to separate the white blood cells from the red, but the methods used in separating these elements are injurious to the cell metabolism (26, 27). . The third method, also in vitro, has been concerned with the metabolism of the leukemic cells obtained from local infiltrations. To our knowledge only one report of this type is available in the literature, namely that of Jackson, Parker, and Glover (13). An analysis of their results shows such marked differences that the only justifiable conclusion is that there is wide variation. Their attack on the problem, however, seems to be the best one, for the mixture of tissues is minimal. The cells may be studied without any possibility of trauma other than that incident to slicing of tissue, and this causes no significant injury, since the numer- ous in vitro investigations of Warburg (31) are corroborated by the in vivo experiments of Tadenuma et d. (29), Cori and Cori (4), and Warburg et al. (32).

The following experiments have been undertaken as part of a comprehensive investigation of transmissible lymphatic leukemia in mice. Studies in the genetics, pathology, and cytology of this disease

1This investigation k a s supported by a grant from the Carnegie Corporation and an appropriation from the Research Fund of Columbia University.

56 1

562 JOSEPH VIOTOR AND MARGARET R. WINTERBTEINER

have been reported by MacDowell, Richter, and Potter (22, 23, 24). The genetically controlled material available for their studies is par- ticularly suitable for metabolic experiments. This report is concerned with metabolic differences between four different lines of transmissible lymphatic leukemia in mice.

EXPERIMENTS &laterial: All animals were of strain C58, in which susceptibility

to the transmitted disease is virtually 100 per cent. They were six to eight weeks old. When the oxygen consumption and glycolysis of normal lymph nodes were measured, the nodes of four animals were combined to obtain sufficient material. In animals inoculated with leukcmia, however, the combined infiltrated nodes of two or three mice were sufficient. I n each case the nodes were from animals of the Name sex and almost invariably litter mates. Necrotic, hemorrhagic, or congested tissues were discarded.

Imculatimg Dose : Four lines of transmissible lymphatic leukemia, designated A, I, M-spleen-D, and M-liver, were used. The inoculating dose was standardized for each line. I n lines I, M-spleen-D, and M- liver the dose was 0.2 C.C. of a suspension made by diluting a thor- oughly minced spleen with 0.9 per cent NaCl, in proportion to its size (2.0 C.C. saline for a spleen 3 x 1 cm.). The mesenteric lymph nodes of line A were suspended in a mixture of 1 C.C. of ascitic fluid and 0.5 C.C. of 0.9 per cent NaCl. Of this suspension, also, 0.2 C.C. was used. Inoculations were intraperitoneal.

Liwes: Line I killed the animals in three and a half to five days, line M-liver in three and a half to six days, line M-spleen-D in four to six days, and line A in six to eight days. Lines M-spleen-D and M-liver are derived from the same case of spontaneous lymphatic leukemia and are called parallel lines, Line M-spleen-D originated in a portion of the Hpleen, whereas line M-liver originated in the liver of the same animal. After twenty-three transfers in which liver was used, line hl-liver was carried with splenic inoculations. Line M-spleen-D waA used for metabolic studies in its sixty-second to seventy-second trans- fer, while line M-liver was used in its fifty-sixth to sixty-third transfer. The lymph nodes of line I were taken three days, of line A five to six days, and of lines M-spleen-D and M-liver four days after inoculation.

The differences in the virulence of the several lines have already been mentioned. These are associated with pathological and cyto- logical differences, Lines I and A have been described by Richter and MacDowell. Line I involves the spleen and all the lymph nodes, and intensely infiltrates the liver and kidneys. Line A is associated with marked peritoneal and slight pleural effusion, very little liver and practically no kidney infiltration, with no involvement of peripheral 11 odes and marked involvement of abdominal and thoracic nodes. Lines M-spleen-D and M-liver show a distribution of lesions similar to line I. These three lines also produce a high white cell count,

STUDIES IN MOUSE LEUKEMIA 563

usually in the hundreds of thousands. With line A, however, there is usually a leukocyte count of 25,000, and a count of 100,000 is rarely reached. Furthermore, in line A, a tumor-like growth develops at the site of inoculation, whereas in lines I and M-liver there is only diffuse infiltration of the abdominal wall ; line M-spleen-D shows more con- gestion than infiltration.

The predominating cell type in line I has been described by Potter and Richter (22). This type is different from that found in lines A and M-spleen-D, but similar to that of line M-liver. The cell types of lines A and M-spleen-D also differ from each other.

It may be stated, then, that the four lines used differ from each other in at least one of the characteristics mentioned, namely, virulence, cell type, distribution, or blood picture.

iVet72od: The oxygen consumption and aerobic and anaerobic gly- colysis were measured with the Fenn modification of the Thunberg differential volumeter. Infiltrated nodes were taken at the time after inoculation when the infiltration of leukemic cells had become so ex- tensive as to obliterate all the normal architecture and transform the node into a mass of leukemic cells. To determine this time, which varies with the line of the cells, special studies were made on the progress of infiltration for lines A, M-spleen-D, and M-liver. In line A only abdominal and thoracic lymph nodes were used; in the other lines the cervical and inguinal nodes were also studied. The nodes were immersed in glucose-free Ringer’s solution and cut into slices 0.2 to 0.3 mm. thick and then weighed to 0.2 mg. on a torsion balance, 30-100 mg. moist weight being placed in each respirometer. The mois- ture content was estimated as 80 per cent, and corrections were made for this factor. Stern and Reincmer (28) report the moisture content of diseased and normal lymph nodes as varying between 69.5 and 80.2 per cent. In work described in a preliminary report (30) the tissues were weighed at the end of the experiment. A series of experiments in which weighings were made before and after the experiment re- vealed constant differences in weights f o r normal lymph nodes and f o r lymph nodes of each individual line of lymphatic leukemia. The values obtained in the previous series are identical with the present series when corrected by this factor. This factor is the average of twelve observations for each line of lymphatic leukemia. Fo r normal lymph nodes the factor is 0.70; for line A, 0.60; and for line I, 0.63. The difference in weight of the tissues before and after the experiment is due to the fact that shaking the respirometer frees the cells from the reticulum on the lymph nodes and they become suspended in the Ringer’s solution.

The oxygen consumption was measured directly with the tissue in Ringer’s solution of 0.9 per cent NaC1, 0.023 per cent KCl, and 0.022 per cent CaCl,, containing 0.2 per cent glucose. I n some cases a phosphate buffer, pH 7.4, was used. The presence of the buffer did not influence the rate or duration of a constant oxygen use. Co? was absorbed by a 0.1N NaOH solution placed in an inset. The glycolysis

564 JOSEPH VICTOR AND MARGARET R. WINTERSTEINER

Days After Date 1 Inoculation Transfer I Qo2 I QZ2 1 Qg& Number

3/25/32 12/22/32 3/30/32 3/30/32 3/31/32 4/1/32

12/22/32 12/23/32 12/23/32 12/24/32 2/21/33 2/21/33

10/31/32 11/17/32 11/18/32 11/19/32 11/23/32 11/25/32 11/28/32 12/6/32 12/5/32 12/6/32 12/7/32

12/7/32

12/9p2

12/10/32

12/7/32

12/9/32

12/10/32 12/10/32

12/21/32 1211 1/32

5 6 6 6 6 6 5

6 5 6

6

Uninoculated (48 Mice) 5.0 7.0 4.7 6.5 5.5 4.4 5.4 5.1 5.4 5.1 5.9 5.4

Inoculated with line A (33 Mice) 159 163 163 163 164 164 165

166 166 166

' 166

243 243 243 243 243 244 244 244 247

7.6 5.9 7.1 6.1 6.2 5.7 6.5 5.9 6.9 6.0 6.5

5.5 5.6 5.8 5.3 5.1 5.5 5.8 5.3 5.3

2.3 3.0 2.0 2.0 1.9 2.1 2.6 1.1 1.1 2.3 2.4 2.7

1.4 2.7 1.5 2.1 1.7 1.7 2.6 2.1 1.5 2.1 1.3

6.8 4.9 6.0 4.4 6.2 5.5 4.6 0.0 5.7

4.4 3.2 3.4 4.7 4.4 4.4 6.3 7.2 8.1 8.0 6.8 8.4

6.9 7.3

11.2 7.3 9.2 6.0 7.8 7.8

10.4 9.8 8.7

17.3 9.5

11.1 11.0 14.6 9.8

11.1 16.1 14.3

was measured by the Warburg method (33). A control respirometer contained tissue immersed in Ringer's solution to which 2.5 x loi2 M NaHCO, was added. The solution was equilibrated with a gas mix- ture of 5 per cent CO, and 95 per cent 0, by bubbling the mixture through it for about half an hour. Then the gas mixture was run through the experimental bottle, after the tissue had been placed in the Ringer-bicarbonate solution for another ten minutes. Flushing of the bottles with the gases was insured by having an outlet through a stop- cock attached to them. The excess aerobic and anaerobic glycolysis was measured with 0.2 per cent glucose in the Ringer-bicarbonate solutions and the tissues and solutions were equilibrated with 5 per cent CO, and 95 per cent 0, for aerobic glycolysis and with 5 per cent CO, and 95 per cent N, for anaerobic glycolysis determinations. The pH in

STUDIES IN MOUSE LEUKEMIA 565

Days After Date 1 Inoculation

Transfer Number 1 Qo* 1 @%a, 1 Q C N 2

the probable error of the differences of the means from the ?ormula P. E.M.~-M.,=.~~ V (P. E.M.~)* + (P. E.M.,)~.

1/23/33 4 1/23/33 4 1/27/33 4 1/27/33 4

1/30/33 4 1/27/33 4

1130133 4 1130133 4 1/19/33 4 1/19/33 4 2/20/33 4 2/20/33 4

Sources of Error: The excess glycolysis measurement by the above method assumes an unchanged respiratory quotient in any of the res- pirometers. Since the control respirometer contains no glucose, the

56 6.9 6.8 23.7 56 6.0 9.8 19.3 57 5.2 8.8 21.2 57 5.7 9.9 19.1 57 5.5 7.7 17.5 58 5.4 9.4 20.1 58 5.6 10.6 21.5 58 6.7 6.0 16.1 55 7.5 7.9 19.4 55 4.9 7.2 18.4 63 4.7 8.1 20.3 63 5.3 8.6 20.7

1/18/33 1/19/33 1/23/33 1/25/33 1/25/33 1/26/33 1/26/33 1/26/33 1/29/33 1130133 2/27/33 2/27/33

4 62 7.2 6.2 11.6 5 62 5.7 4.6 14.3 5 63 6.9 7.1 16.1 4 64 6.8 6.7 15.9 4 64 7.4 6.4 16.3 4 64 8.1 6.9 15.5 4 64 7.3 5.7 16.3 4 64 6.4 7.2 15.2 4 65 7.1 6.5 16.4 4 65 6.4 5.6 15.1 4 72 5.4 5.6 15.1 4 72 5.4 5.3 15.9

566 JOSEPH VIOTOR AND MARGARET R. WINTERSTEINER

NORMAL LINE A LINK I M . Liver

M .Sploen.D I I I

b I 2 4 6 a cb

AEROBIC iGLvcOLVSlS Qoa -2

LINE A LINE I

I I 8 I I I I I I I 0 12 I4 I6 i I8 zb

AN~R0WC4GLYCO?&IJ 8& lo

WI,. 1. METABOLIC DIYFE~ENCES BETWEEN THE LYMPH NODES OF NORMAL MICE OF STRAIN C58 AND THOSE INOCULATED WITH FOUR DIJTEEENT LINEE OF LYMPHATIO

LEUKEMIA, A, I, M-LI-, AND M-SPLEEN-D Mean values are given by total lengths of bars; open ends indicate 1 x probable error of

each mean.

to decrease the oxygen consumption of all the leukemic and normal lymphoid tissues we have studied.

RESULTS In Table I are recorded data of observations, interval between in-

oculation and takiiig the tissue, the number of transfer generations and the values of the individual metabolic determinations for normal nodes and those infiltrated by each of the lines. Table I1 gives mean values, standard deviations, and probable errors of the means. Table 111 shows the differences between the means for normal nodes and those infiltrated with the various cell lines, as well as differences between cell lines ; probable errors of these differences are given with the ratio of the difference to its probable error. When this ratio is 3 or over, it is usually considered statistically significant ; those over 4 are italicized.

STUDIES IN MOUSE LEUKEMIA 567

' 5.78 f .35 1.80 8.31 f .33 1.64

12.76 f .60 2.68 19.78 f .36 1.84 15.31 f .24 1.25

Fig. 1 is a graphic representation of the means and their probable errors.

There are marked metabolic differences between normal and leu- kemic lymph nodes and also between nodes of mice inoculated with dif- ferent lines of leukemic cells. Lines A and M-spleen-D have higher rates of oxygen consumption than normal lymphoid tissue. The

TABLH~ 11: Average Rate of Oxygen Consumption and Aerobic and Anaerobic Glywlysis of Normal Lyrn,ph Nodes and. Those Infiltrated with Different Lines of Lymphatic

Leukemia (Mice of Strazn C58)

2.13 f .10 1.88 f .10 5.57 f .16 8.40 f .26 6.15 f .15

Normal Line A Line I M-liver M-spleen-D

.53

.47

.73 1.32 .76

QOZ

- 2 5 f .14 1.8 +Y.44 f .20 17.2 +627 f .31 20.2 +Q.OB f .18 22.4

+3.69 f .19 19.4 +6.5.2 f 28 23.3 +427 f .18 23.7

+8.83 f .30 9.4 + .58 f .22 2.6

-2.25 f .30 7.5

Mean

+ 6.65 f .48 5.3 + 6.98 f .67 10.4 +ld.OO f .48 29.2 + 9.53 f .38 25.1

+ 4.45 f .68 6.5 +f l .47 f .49 23.4 + 7.00 f .41 14.3

+ 7.0.2 f .70 10.0 + 6.66 f .65 3.9

- 4.47 f .43 10.4

5.45 f .14 6.40 f .ll 5.47 f .03 5.78 f .16 6.68 f -15

Stan. Dev.

.70

.56

.14

.83

.76

_____ Mean I St,an. Dev. I Mean, 1 Stan. Dev.

TABLE 111: Metabolic Differences between Lymph Nodes of Normal Mice of Strain C58 and Those Inoculated with Lines of Lymphatic Leukemia; Differences between Lines

(Statistically Significant Differences are Italicized)

COMPARED WITH NORMAL: Line A Line I M-liver M-spleen-D

Line I M-liver M-spleen-D

M-liver M-spleen-D

M-spleen-D

COMPARED WITH LINE A:

COMPARBD WITH LINE I:

COMPARED WITH LINE n LIVER :

Diff Diff. - 1'. I2

+ .96 f . l6 5.9 + .u2 f .12 0.2 + .33 f .19 1.7 +I23 f .10 6.5

- .93 f .ll 8.4 - .02 f .19 3.3 + .28 f .19 1.5

+ .31 f .16 1.9 +f.62 f .15 8.1

+ .80 f .2L 4.1

'i'ne metaDoiic ainerences uetween L I I ~ I L I I ~ S are Bummarixea in Table 111. Comparison of line A with the other lines shows its rate of oxygen consumption to be similar to that of M-spleen-D but greater than that of either I or M-liver. Its rates of aerobic and anaerobic glycolysis are less than in the other lines.

568 JOSEPH VICTOR AND MARGARET R. WINTERSTEINER

When the metabolism of line I is compared with that of the other lines, it is also found to differ from them. Its rate of oxygen consump- tion is less than that of M-spleen-D but similar to M-liver. On the other hand, the aerobic glycolysis of line I is similar to M-spleen-D, but less than M-liver, while its anaerobic glycolysis is less than that of the other lines.

, Comparison of lines M-liver and M-spleen-D, derived from different organs of the same spontaneous case, shows them to differ metabolically. The rate of oxygen consumption of line M-liver is less than that of M- spleen-D, while the rates of both aerobic and anaerobic glycolysis are less than in the latter.

The aerobic and anaerobic e'nergy production of t h e leukemic cells i s greater than normal. The leukemic cells may have higher rates of oxygen consumption than normal (lines A and M-spleen-D), or of aerobic glycolysis (lines I, M-liver, and M-spleen-D). However, the total aerobic energy production derived from oxygen consumption and glycolysis is greater in the leukemic than in the normal cells. The anaerobic glycolysis of the leukemic cells studied is always greater than that of normal cells.

Although the metabolic differences between normal and leukemic cells are in the direction of increased energy production by the leukemic cells, each line shows a separate deviation from normal. This is judged by rates of oxygen consumption and aerobic and anaerobic glycolysis. It is true whether the cell lines have been derived from the same organ of different spontaneous cases (A, I, and M-spleen-D) or from different organs of the same spontaneous case (M-liver and M-spleen-D).

DISCUSSION The experiments on the parallel lines, i.e. those derived from the

Same case of spontaneous lymphatic leukemia, were done alternately. The dates in the tables show that observations on both lines were often made on the same day. These differences, therefore, are not due to variations involved in repetition of experiments. Although the experi- ments with lines A and I were not carried out alternately, the last ex- periment with line A was made on the same day as the first with line I. The tlifferences observed also seem to rule out the possibility of varia- tions due to causes mentioned above, especially since the gases, solu- tions, respirometers, and temperature were the same.

The uniformity of the results in these experiments is striking when compared with results of similar experiments reported in the litera- ture. Warburg's figures (31) for nineteen observations on the Flex- iier-Jobling rat carcinoma show a Qol of 7.2 with a standard deviation of 3.2,45 per cent of the mean. When these figures are compared with the above results with normal lymph nodes, lines A, I, M-spleen-D, and M-liver, where the standard deviation of Qolis 10, 9, 3, 12, and 14 per cent of the mean respectively, one cannot help feeling that many factors are responsible for such variations in attempts at repetition.

STUDIES I N MOUSE LEUKEMIA 569

No mention is made by Warburg of the age of the animals used in his experiments, nor of the relative susceptibility of the hosts. In our experiments, where susceptibility to the transmission of the disease is genetically controlled and the age of the host is likewise uniform, these possible variants are minimal.

In the same paper Warburg reports the Q02, Q;h2,. and of n normal human lymph gland as 3.8, 2.2, and 4.7, respectively. The Q,,? value is lower than any of our observations on the lymph nodes of over 300 mice, six to eight weeks old, with or without leukemia. The glycolysis values are similar to our normal values, however. The fonr observations on the Qo2 of normal lymph nodes of Jackson, Parker, and Glover show variations from 0.6 to 11.8. Their nodes from cases of lymphoma, which include lymphosarcoma, lymphatic leukemia, lym- phocytoma, lymphoblastoma, and reticulum-cell sarcoma, including twelve cases, show variations from 0.7 to 12.6 in QO2, with a mean calculated from their protocols of 5.8 and a standard deviation of 4.1, 71 per cent of the mean. Their glycolysis observations likewise showed marked variations. The method used for determining aerobic gly- colysis, namely the disappearance of glucose from the nutrient fluid, does not distinguish the glucose that is oxidized from that which is fermented. Furthermore, many different cell types are included in their group of lymphomata. The authors do not say anything about the concentration of glucose in the nutrient fluid in their experiments. This has been shown by Warburg (31) and Levene and Bieyer (17) to be an important factor in the rate of glycolysis.

T h e energy requirements of the cells in transmissible lymphatic leukemia in the mouse are greater than those of normal lymphoid tissue. If the oxygen consumption is not increased, then the aerobic glycolysis is increased. In one case, line B1-spleen-D, both were increased. I n no case was there a decrease in oxygen c o n s u ~ ~ ~ ? ~ i o n . This is quite con- tradictory to the interesting theory elaborated by Warburg (31), that when the respiration of a cell is injured and the cell has the power of glycolysis, it will assume this type of metabolism and so become neo- plastic. A good part of the evidence for this lies in the low oxygen consumption of malignant tumors such as the Flexner-Jobling rat carcinoma and many of the adenocarcinomas and basal-cell and squa- mous-cell carcinomas of man when compared with liver and kidney epithelial cell metabolism. However, the metabolism of the Flexner- Jobling carcinoma, originating in a rat’s epididymis, has never been compared with the metabolism of normal epididymis epithelium. Fur- thermore, the metabolism of normal squamous, basal, and acinar epi- thelium of various types lias not been compared with that of tumors derived from the organs containing those cell types. The experiments described in this report are the only ones with which we are familiar, where a comparison of the qetabolism of normal and malignant cells of the same type is made.

The very suggestive experiments of nickens and Simer ( 7 ) seem

In the experiments reported here, one fact becomes apparent.

570 JOSEPH VICTOR AND MARGARET R. WINTERSTEINEB

to throw more light on the nature of the glycolysis mechanism in tu- mors, namely that tumor cells do not oxidize lactic acid. Whether the inability to oxidize lactic acid is a factor in its production in large quantities in these leukemic tissues is yet to be determined.

The presence of glucose in Ringer’s solution does have a depressant effect upon the oxygen consumption of both the normal and leukemic lymph nodes. This loss of energy from oxidation seems to be some- what compensated by glycolysis ensuing after the addition of glucose. This observation will be the subject of a further communication.

There seems to be no fixed correlation of the metabolic changes with the virulence of the disease, as judged by the interval between inoculation and death of the host, when the metabolism of different lines is compared. Line I, for example, has a shorter interval than line M-liver, yet its Q$, and QEbS are less than those of M-liver. The same applies to line M-liver and M-spleen-D. The rate of growth of the cells does not seem to be a metabolic factor either. The greatest proportion of dividing cells is found in line I, yet its metabolism is lower than that of both line M-liver and M-spleen-D. A further factor that might be correlated with metabolism is the morphology of the cell. Yet although the cell type in lines I and M-liver is the same, the metabolism is different. This suggests that there are inherent meta- bolic differences between the different lines of transmissible lymphatic leukemia which may obscure the relationships between metabolism and other characteristics.

Another question arises : Does leukemic tissue have a metabolism similar to tumor metabolism4 Warburg (33) states that the ratio of aerobic glycolysis to oxygen consumption in malignant tumors is 3, while that of benign tumors is about 1. Other observers have found that many sarcomata do not have as high an aerobic glycolysis-oxygen consumption ratio as those reported by Warburg and others for car- cinomata (13). For line A this ratio of Q&/Qo, is 0.3 ; for line I it is 1.0; for line M-spleen-D, 0.9, and for line M-liver, 1.5. Most of the lines, then, resemble benign tumors according to his criteria. How- ever, the fact that certain pathological overgrowths ( 5 ) are associated with a metabolism similar to this, makes it impossible to differentiate infections from neoplastic tissue, using oxygen consumption . and aerobic and anaerobic glycolysis as criteria.

S u MMARY

1. The oxygen consumption, aerobic glycolysis, and anaerobic gly- colysis of lymph nodes of normal mice and of those inoculated with different lines of transmissible lymphatic leukemia were measured in animals in which age and genetic constitution were controlled,

2. There are metabolic differences between normal and leukemic lymph nodes.

3. Inherent metabolic differences were observed not only between different lines of leukemia derived from the same organ in different

STUDIES IN MOUSE LEUKEMIA 57 1

spontaneous cases, but also between lines derived from different or- gans in the same spontaneous case.

4. The metabolism of the individual line of transmissible leukemia was consistent during the period of observations.

5. In no case was the oxygen consumption of leukemic nodes less than normal; in some cases it was greater than normal.

6. The aerobic glycolysis of leukemic lymph nodes was usually greater than normal. The anaerobic glycolysis was always greater than normal.

7. The results are statistically compared with others reported in the literature. Factors causing variations in results are discussed.

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