An Appraisal of a Biological Response by Strain HeLa
Cells in Tissue Culture to 100 Human Sera*
GEORGEG. RosEf
(Division of Biology, University of Texas M. D. Anderson Hospital and Tumor Inst., and Tissue Culture Laboratory,
Hermann Hospital, Houston So, Texas; and The Tissue Culture Laboratory, The University of Texas,
Medical Branch, Galveston, Texas)
Any means by which human serum may be
characterized, be it physical, chemical, or biologi
cal, offers a potential diagnostic method. The work
presented herein, developed along the biological
approach, is not offered as a diagnostic test, but
merely as a summation of semi-quantitative ob
servations on the behavior of strain HeLa cells
(5-9, 11, 24-27, 29, 30) which were influenced by
human serum.
In seeking a biological response of cells to serum,
the VP cells (variant pinocytic cells) were ob
served (24-27, 30). These cells differed from other
cells of HeLa cultures viewed through a phase
contrast microscope (Bausch and Lomb) in that
their cytoplasm contained a varying number of
optically dense (black) spherical granules (4-25 ju).
Preliminary studies showed that VP cells were
seldom seen in HeLa colonies cultivated in any
of the many conventional standard nutrients con
taining little or no human serum and only 20 per
cent animal serum. After such a standard nutrient
was removed and replaced by a 100 per cent
human serum, a maximum number of VP cells
occurred in 24 hours. The VP response has been
discussed in greater detail in other publications;
however, it may be of some value to add that it
is due to pinocytosis (Figs. 1, 2) or drinking (2,
5-11, 13-20, 22, 24-27, 29-31) by the HeLa cells.
It seemed reasonable to believe that a system for
serum analysis might be devised, since (a) HeLa
cells could be cultivated in multipurpose culture
chambers (21, 23, 28) as round colonies, (6) the
VP cells occurred most prominently around the
periphery of these colonies, (c) these colonies could
be viewed with phase contrast (1) or interference
contrast (1, 28) microscopes without special prep* This work was supported by a grant-in-aid from the
American Cancer Society and by a grant-in-aid from the Amer
ican Cancer Society administered by Dr. C. M. Pomerat.
t Mailing address: Tissue Culture Laboratory, Hermann
Hospital, Houston 25, Texas.
Received for publication October 23, 1957.
arations, and (d) the number of VP cells had
been found to vary in colonies cultivated in serum
from different persons. Such an analysis based on
the 24-hour maximal VP response to serum speci
mens obtained at random from 100 humans is
recorded herein.
MATERIALS
AND METHODS
Stock cultures of strain HeLa were maintained by conven
tional roller tube technics. The HeLa cells were subcultured
from the roller tubes to multipurpose culture chambers in a
manner indicated in earlier reports (21, 23, 28). The 1-mm.sized subcultured fragments were cultivated directly upon the
glass walls of the chambers and were nourished semi-weekly
with a nutrient fluid composed of Medium No. 1066 (Connaught Medical Research Laboratories, a variation of Medium
No. 858) (12), 75 per cent; calf serum, 20 per cent; and whole
egg ultrafiltrate (4), 5 per cent. Occasionally, the nutrient was
fortified with 5 per cent pooled human serum. Potassium peni
cillin (Squibb) was added to the nutrient at a level of 1000
units/cc. This nutrient did not provoke a VP response. After
the colonies had been cultivated so that their diameters had in
creased to approximately 4 or 5 mm. (7-10 days), the nutrient
fluid was removed, and the 100 per cent human serum to be
tested was added to the chambers. The blood for this serum
was (a) obtained the day before the testing period, (6) left in
serum bottles for 1-2 hours at room temperature, (c) re
frigerated overnight at 5°C., and then (d) re-incubated 1-2
hours at 37°C. The serum was poured from the bottles into
tubes which were centrifuged at 1000 r.p.m. for 15 minutes.
The clear supernatant serum was removed with an 18-gauge
needle and placed in polyethylene bottles. Three or four cham
bers containing one HeLa colony each were selected to analyze
each person's serum sample. After the serum was added to the
chambers (2 cc/chamber), the cultures were incubated in the
horizontal position for 24 hours. One or 2 hours before micro
scopic observations were made, the chambers were placed in a
vertical position which rendered the microscopic field clear and
free from cellular or serum debris. Each chamber was surveyed
with a Bausch and Lomb phase contrast binocular microscope
through the 21X objective and 10X wide field oculars. The
observer did not know the number of the patients' serum nor
the patients' diagnoses. The chambers were moved so that only
the periphery of each culture was observed, and the VP cells
(Fig. 2) within this circular field of view were counted only one
time. Two diameters of the colony were obtained grossly with
a millimeter rule, and an average was determined. This dia
metric measurement was recorded as a complementing figure
to the VP count. The values for three or four chambers of each
serum were totaled and averaged. The average colony diameter
411
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TABLE 1
VITALSTATISTICS,DIAGNOSIS,
ANDVP INDEXOF100PATIENTS
Age, Color, Sex
53 WM
58 CM
51 WM
69 CM
77 WM
CáWM
48 CM
56 WM
63 WM
72 WM
58 WM
61 WM
71 WM
54 WM
73 CM
52 CM
75 WM
39 WM
70 WM
67 WM
65 WM
66 WM
40 WM
17 WM
68 WM
50 WM
38 WM
66 WM
44 WM
41 CM
86 WM
47 WM
79 WM
61 WM
78 WM
63 WM
46 WM
72 CM
81 WM
55 WM
72 CM
20 WM
21 CM
38 WM
72 WM
43 WM
65 WM
74 WM
68 WM
30 WM, 29 CM
52 WM, 38 CM
29 CM, 34 CM
51 WF
36 CF
69 WP
69 WF
41 CF
78 WF
55 CF
50 CF
65 CF
43 CF
45 WF
32 CF
64 WF
HI WF
74 WF
58 WF
60 WF
27 CF
85 WF
44 WF
40 WF
Diagnosis
Adenocarcinoma of the kidney, carcinoma cells in ascitic fluid
Metastatic adenocarcinoma, lymph node biopsy
Squamous-cell carcinoma of the lung
Adenocarcinoma of the prostate, diabetes mellitus
Adenocarcinoma of the colon
Bronchiogenic carcinoma, epidermoid
Carcinoma of the tongue
Adenocarcinoma of the colon
Adenocareinoma of the colon
Adenocarcinoma of the prostate
Since 1950 metastatic melanoma
Squamous-cell carcinoma of the hand
Squamous-cell carcinoma of the esophagus (gr. Ill)
Carcinoma of the larynx
Undifferentiated squamous carcinoma
Metastatic squamous carcinoma (gr. Ill)
Squamous carcinoma of retro-molar trigone (gr. Ill)
Hodgkin's disease (2 years), extensive x-ray
Squamous carcinoma of the skin on the hand
Neck tumor, unclassified squamous carcinoma (primary lung or
pharynx)
Multiple basal and squamous-cell carcinoma of the face and neck
Degen, joint disease, osteoarthritis, arterioscl., diverticulosis (colon)
CNS syphilis, lobar pneumonia
Concussion of the brain, multiple trephinations
Appendectomy, abcess
Gunshot wound, right foot
Internal hemorrhoids, anal fissure
Diabetes mellitus, paralysis agitans, eczema, arterioscl. ht. disease
Lobar pneumonia
Heart disease, undiagnosed
Infectious eczematoid dermatitis, arterioscl., senile emphysema
Internal and external hemorrhoids
Arterioscl., dementia
Arterioscl., dementia
Hypertension, cardiovascular accident (3 years), acute dementia
Chronic ostemyelitis of the femur
Chronic granuloma tous inflara, right lung (probably TB)
Prostatism
Arterioscl. ht. disease, diabetes mellitus, prostatic hypertrophy,
emphsema
Amputation of toe following trauma
CNS syphilis, cirrhosis of the liver
Acute lumbrosacral strain, epileptic
Pneumonia, hemolytic staph.
Abdominal pain functional or undiagnosed
Chronic inflam. bladder
Thyroiditis (clinical diagnosis)
Cutaneous horn on the nose
Peptic ulcer
Biopses, seborrheic keratosis left ear
"Normal" males. Range of VP Index, 221-356
Adenocarcinoma of the ovary
Carcinoma of the cervix (radium Rx)
Malignant melanoma
Glioblastoma multiforme (brain biopsy)
Carcinoma of the cervix (radium Rx), carcinomatosis
Carcinoma of the stomach, neoplastic colon polyp
Carcinoma of the cervix
Carcinoma of the cervix (radium Rx)
Leiomyosarcoma of uterus
Carcinoma of the cervix (radium Rx)
Granulosa-cell carcinoma of the ovary, cylindrical type
Carcinoma of the cervix in situ
Low grade follicular carcinoma of the thyroid, Hasimato's disease
Carcinomatosis of the abdomen, carcinoma cervix
Adenocarcinoma of the colon
Adenocarcinoma of the thyroid
Adenocarcinoma of the pancreas
Chronic cervicitis
Fractured femur
Diabetes mellitus
Functional uterine bleeding
Vf index
849
569
465
635
402
245
507
433
406
324
325
331
399
241
200
248
350
365
394
439
886
64
186
408
365
621
425
840
709
468
294
168
210
263
277
371
492
207
166
297
280
223
157
280
313
341
230
155
522
288
247
516
200
347
495
235
319
223
335
260
377
126
269
175
494
459
272
180
153
260
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ROSE—StrainHeLa Cell Response to Human Sera
TABLE
Age,Color,Sex
46 WP
17 WF
45 WF
32 WF
24 WF
76 WF
26 WF
41 CF
48 CF
17 WF
55 CF
34 CF
36 WF
35 WF
21 WF
23 WF, 24 WF,
29 WF, 30 WF,
30 WF, 34 WF,
29 WF, 44 WF,
25 WF
1—Continued
Diagnosis
VP index
Abd. hysterectomy, eervicitis, endometrial dysplasia
Acute appendicitis
Myoma uteri and menorrhagia
Colloid adenoma with cystic degen. of the thyroid
Cervicitis, chronic
Chronic cholecystitis, early Laennec's cirrhosis, biliary obstruction
Incisional hernia
Atypical squamous epithelial hyperplasia of cervix, leiomyoma uteri
Nontoxic nodular goiter
Functional abdom. symptoms
Hypertension
Toxic thyroid, thyroidectomy
Hemorrhoidectomy
Hemorrhoidectomy
Chronic dislocation of the patella, tendon transplant
"Normal" females. Range of VP Index, 214-484
for each serum was multiplied by TTto obtain the average
peripheral measurement. This was divided into the average
number of VP cells, the quotient multiplied by 100, and the
result termed the "VP Index." The patients used in this anal
ysis were obtained at random from in-patients, out-patients,
and personnel of the Hermann Hospital, clinic, and hospital
patients of The University of Texas M. D. Anderson Hospital
and Tumor Institute, Houston, Texas, as well as from visitors
to this laboratory.
RESULTS
A comprehensive listing of the 100 subjects
whose serum was analyzed is shown in Table 1.
For convenience only, the males were listed before
the females. Within each of these sex groups, the
cancer patients were listed first; the pathological
patients second; and those whom we considered
to be normal, since they were in a nonpatient
category, third. Each individual was identified by
age, sex, racial color, diagnosis, and the VP Index
of the serum specimen obtained.
The average VP Index for all 100 persons tested
is shown in Table 2. The 38 cancer patients were
averaged to have an index of VP/366, the 47 path
ological patients VP/300, the fifteen normals
VP/299, the 62 controls (pathological plus normal)
VP/300, and the total 100 individuals tested
VP/325. It was assumed that the VP Index for
the control group (VP/300) was a critical value,
and the second part of Table 2 indicates the
percentage of patients in each of the four cate
gories who had a value above VP/300. Twenty-six
of the 38 (68 per cent) cancer patients, nineteen
of the 47 (40 per cent) pathological patients, four
of the fifteen (27 per cent) normals, and 23 of
the 62 (37 per cent) controls had a VP Index above
this critical value.
The average VP Index for the male group is
shown in Table 3. Twenty-one cancer patients had
413
364
348
322
229
166
246
119
225
472
221
95
248
371
338
667
307
TABLE 2
SUMMARY
VPINDEXDATADERIVED
FROMTABLEl
I. Average VP Index for all persons tested:
Cancer patients (38)*
VP/366
Pathological patients (47)
VP/300
Normal (nonpatient persons) (15)
VP/299
Control group (#2 plus #3) (62)
VP/300
Total individuals tested (100)
VP/325
II. Individuals above the control critical
value of VP/300:
Cancer patients (26/38) f
68 per cent
Pathological patients (19/47)
40 per cent
Normals (4/15)
27 per cent
Controls (23/62)
37 per cent
* Figures in parentheses indicate number of per
sons analyzed.
t Figures in parentheses indicate fraction of pa
tients above critical value.
TABLE 3
MALEVP INDEXDATADERIVEDFROMTABLEl
I. Average VP Index for male group:
Cancer patients (21)*
VP/405
Pathological patients (28)
VP/315
Normal (nonpatient persons) (6)
VP/288
Control group (#2 plus #3) (34)
VP/310
All males tested (55)
VP/347
II. Males above the control critical
value of VP/310:
Cancer patients (17/21)f
81 per cent
Pathological patients (12/28)
43 per cent
Normals (2/6)
34 per cent
Controls (14/34)
41 per cent
* Figures in parentheses indicate number of per
sons analyzed.
t Figures in parentheses indicate fraction of pa
tients above critical value.
an average index of VP/405, the 28 pathological
patients VP/315, the six normals VP/288, the 34
controls (pathological plus normal) VP/310, and
the total 55 males tested VP/347. Here again we
considered the control group to be the critical
value (VP/310) and compared each of the four
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414
Vol. 18, May, 1958
Cancer Research
categories with this value. Seventeen of the 21 (81
per cent) cancer patients, twelve of the 28 (43
per cent) pathological patients, two of the six (34
per cent) normals, and fourteen of the 34 (41 per
cent) controls had a VP Index above this critical
value of VP/310.
The average VP Index for the female group is
shown in Table 4. The seventeen cancer patients
TABLE 4
FEMALE
VPINDEXDATADERIVED
FROMTABLEl
I. Average VP Index for female group:
Cancer patients (17)*
VP/318
Pathological patients (19)
VP/279
Normal (nonpatient persons) (9)
VP/307
Control group (#2 plus #3) (28)
VP/288
AU females tested (45)
VP/299
II. Females above the control critical
value of VP/288:
Cancer patients (9/17) f
53 per cent
Pathological patients (7/19)
37 per cent
Normals (3/9)
33 per cent
Controls (10/28)
36 per cent
* Figures in parentheses indicate number of per
sons analyzed.
t Figures in parentheses indicate fraction of pa
tients above critical value.
these three cancer groups for male, female, and
male plus female categories. In all three cancer
groups the male and the male plus female cate
gories are greater than the female. The control
group VP Indices (Critical Values) shown in
Tables 2-4 are also included. Part II contains
percentage values for the three cancer groups of
the different sex categories which relates the num
ber of patients with VP Indexes above their respec
tive control critical value. Seven of seven male
(100 per cent) and two of the six female (33 per
cent) adenocarcinoma patients were above their
respective control critical values. Eight of the
twelve male (67 per cent) and five of the eight
female (63 per cent) squamous-cell carcinoma pa
tients were above their respective control critical
values. Two of the two male (100 per cent) and
two of the three female (67 per cent) sarcoma
patients were above their respective control criti
cal values.
A comparison of the average VP Indices for all
patients who were in the hospital versus those who
were in the laboratories or clinics when their blood
was drawn is shown in Table 6. The cancer patients
TABLE 5
VPINDEXDATARELATING
ADENOCARCINOMA,
SQUAMOUS-CELL
CARCINOMA,
ANDSARCOMA
VARIANCES
TOTHESEXES
Males
Females
I. Average VP Index:
(7)* VP/445
Adenocarcinoma patients
(6) VP/289
Squamous-cell carcinoma patients
(12) VP/392
(8) VP/349
Sarcoma patients
(2) VP/345
(3) VP/293
Control group (critical value)
(28) VP/288
(34) VP/310
(from Tables 2-4)
n Individuals above control critical value:
Adenocarcinoma patients
(2/6) 33 per cent
(7/7)t 100 per cent
Squamous-cell carcinoma patients
(8/12)
67 per cent
(5/8) 63 per cent
(2/2)
—
Sarcoma patients
100 per cent
(2/3) 67 per cent
* Figures in parentheses indicate number of persons analyzed.
f Figures in parentheses indicate fraction of patients above critical value.
had an average index of VP/318, the nineteen
pathological patients VP/279, the nine normals
VP/307, the 28 controls (pathological plus normal)
VP/288, and the total 45 females tested VP/299.
The control group was also considered to be the
critical level for the female category, and the four
groups were compared with this value. Nine of
the seventeen (53 per cent) cancer patients, seven
of the nineteen (37 per cent) pathological patients,
three of the nine (33 per cent) normals, and ten
of the 28 (36 per cent) controls had VP Indexes
above the critical value of VP/288.
The cancer patients' VP Indices were further
segregated into adenocarcinoma, squamous-cell car
cinoma, or sarcoma, as shown in Table 5. Part
I of this table contains the average VP Index of
Males plus females
(IS)
(20)
(5)
(62)
VP/365
VP/365
VP/314
VP/SOO
(9/13) 69 per cent
(13/20) 65 per cent
(4/5) 80 per cent
who were in the hospital (18) had an index of
VP/341, whereas those (20) who had their blood
drawn either in the clinics or in the laboratories
had an index of VP/389. The controls who were
in the hospital (41) had an index of VP/302,
whereas those (21) who had their blood drawn
either in the clinics or in the laboratories had an
index of VP/296.
A comparison of the average VP Indices of
colored versus white persons is shown in Table 7.
The colored cancer patients (12) had an index of
VP/396, and the white cancer patients (26) had
an index of VP/353. The colored controls (13) had
an index of VP/266, and the white controls (49)
had an index of VP/309.
The average VP Index of three age groups is
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ROSE—StrainHeLa Cell Response to Human Sera
shown in Table 8. Of the total group analyzed
(100) those who were less than 30 years of age
(16) had an index of VP/287, those who were 30
to 60 years of age (48) had an index of VP/349
and those who were more than 60 years of age
(36) had an index of VP/310. Of the cancer
patients (38) analyzed there were no patients less
than 30 years of age; those who were 30-60 years
of age (19) had an index of VP/380, and those
who were more than 60 years of age (19) had an
index of VP/352, In the control group (62) those
who were less than 30 years of age (16) had an
index of VP/287, those who were 30-60 years of
age (29) had an index of VP/329, and those who
were more than 60 years of age (17) had an index
of VP/263.
To obviate the factor of the effect of food intake
we tested fasting samples from three healthy males
versus their postprandial samples (with high
TABLE6
VPINDEXDATAGROUPED
ACCORDING
TO
PHYSICAL
STATUS,
WELL-BEING
AND
ENVIRONMENT
OFPATIENTS
I. Average VP Index for all patients
hospitalized when blood drawn:
Cancer patients (18)*
VP/341
Controls (41)
VP/302
II. Average VP Index of all persons
whose blood was obtained in the
the clinic or laboratories:
Cancer patients (20)
VP/389
Controls (21)
VP/296
* Figures in parentheses indicate number of per
sons analyzed.
415
group average used as the critical discriminating
level, 68 per cent of the cancer patients and 37
per cent of the control group had a VP Index above
this level. According to the principles and criteria
set forth by Drs. Dunn and Greenhouse (3), at
least 90 per cent of the cases with early localized
cancer should show a positive reaction and there
should be no more than 5 per cent false positives.
These results then fall far short of such an ideal
value. In the evaluation of the males (Table 3),
81 per cent of the cancer group was above the
control critical value and, therefore, somewhat
closer to the theoretical ideal. Within the female
TABLE 7
VP INDEXDATAGROUPED
ACCORDING
TO RACE
Average VP Index of colored versus
white persons:
Colored cancer patients (12)*
VP/396
Colored controls (13)
VP/266
White cancer patients (26)
VP/353
White controls (49)
VP/309
*Figures in parentheses indicate number of
persons analyzed.
TABLE 8
VPINDEXDATAGROUPED
ACCORDING
TOAGE
cream) and found that the food intake did not
vary the VP response. Also, serum from one male
and from one female of the "normal" groups
re-evaluated after 6 months were found to have
VP Indexes very close to their original values.
I. Total group analyzed (100):
Less than 30 years (16)*
VP/287
Thirty to 60 years (48)
VP/349
Greater than 60 years (36)
VP/310
II. Cancer patients (38):
Less than 30 years (0)
Thirty to 60 years (19)
VP/380
Greater than 60 years (19)
VP/352
III. Controls (62):
Less than 30 years (16)
VP/287
Thirty to 60 years (29)
VP/329
Greater than 60 years (17)
VP/263
* Figures in parentheses indicate number of
persons analyzed.
DISCUSSION
A quick review of the tables illustrates why this
work is not presented as a diagnostic test for
cancer but merely as a summation of semiquantitative biological observations on strain HeLa cells which were influenced by human serum.
Even if the results had been sharply defined, the
technic would not be applicable to a major testing
program. Since the numerical results did not ap
proach suggested desired values for cancer diag
nostic tests, they were not subjected to a statistical
evaluation.
Certain trends among groups of persons are
indicated in Tables 2 through 8. In Table 2, a
summation of all 100 persons, it is noted that the
cancer patients had on the average a higher VP
Index than the control group. With the control
group (Table 4) the cancer patients were not as
markedly discriminated by the average control VP
Index level. Noting that the females on an average
had a lower VP Index within both the cancer and
the control groups than did the males suggested
a possible sex hormonal influence upon this re
sponse. It was interesting to observe that, in Tables
2-4, the control groups which were selected as the
critical level were truly the most discriminating
point. Further, it was noted that the so-called
pathological and normal patients had a strikingly
close average VP Index. The point here is that,
even though there were a number of false positive
tests, there were not a great many more within
the pathological group than within the normal
group. Of all 100 persons the average VP Index
for the pathological and normal groups was es-
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416
Cancer Research
Vol. 18, May, 1958
sentially identical, although the percentage of
pathological patients who had a value above
the control discriminating level was somewhat
higher (13 per cent) than that of the normal
group.
An attempt to break the data down into lesser
cancer categories (Table 5) demonstrated why the
male group had a higher percentage of cancer
patients above the critical value (Table 3) than
did the females (Table 4). All seven of the male
adenocarcinoma patients had a VP Index above
the critical value, in contrast to only two of the
six (33 per cent) female adenocarcinoma patients.
Male and female squamous-cell carcinoma patients
had equivalent percentages above their control
critical values and, therefore, had no contrasting
influence upon the total cancer patient analyses
for each group. The sharp distinction between the
two sexes in the adenocarcinoma category may
be related to the fact that HeLa cells were derived
from a female squamous-cell carcinoma. It is in
teresting then to speculate on the occurrence of
converse results had strain HeLa cells originated
from a male. Similarly, a strain of cells derived
from an adenocarcinoma might favor a high VP
Index in a squamous-cell serum category of the
opposite sex.
To determine the effect of other more or less
constant factors, the average VP Indices were
re-grouped in Tables 6 through 8. In Table 6 it was
shown that the hospitalized cancer patients had
a lower average VP Index than those cancer
patients whose blood was obtained in the labo
ratories or clinics. The controls in either case were
approximately the same. This may indicate that
the technic is more sensitive for early cancer
patients than for those with far advanced cases.
The average VP Index for the cancer patients of
both the colored and white races (Table 7) was
higher than the average VP Index for both the
colored and white controls. It was interesting to
observe that the colored persons had a higher
average VP Index within the cancer groups and
a lower average VP Index within the control group
and, therefore, demonstrated a broader spread of
values than the white persons. The total number
of persons evaluated was broken down into three
age groups (Table 8), and it is immediately obvious
that a very important age group was not evalu
ated. This was the cancer group under 30 years
of age. As indicated in the section on "Materials
and Methods," samples were drawn from persons
immediately available, and this group did not fall
into our category of availability. We can see,
however, that within the 30- to 60-year age group
the average VP Index for the cancer patients was
higher than the average VP Index of the greaterthan-60-year age group. The average control VP
Index of the 30- to 60-year age group was higher
than (a) the less-than-30- and (b) the greaterthan-60-year age groups. It might be expected,
therefore, that the average VP Index for cancer
patients less than 30 years of age would also be
lower than the average VP Index of cancer patients
in the 30- to 60-year age group. This indicated
once again that, if values of this sort are obtained
from a larger population by a revised and more
sensitive technic, the results obtained would have
to be scaled according to the type of information
shown in Tables 6-8.
The VP phenomenon analyzed in this report
was basically an ingestivo action of cells provoked
by the serum in which they were bathed. Since
only peripheral cells having free membrane borders
are the most energetic VP cell-responders to the
serum environment, a method which will utilize
many dispersed and, therefore, potential VP cells
is contemplated in an effort to increase the sen
sitivity of this type of examination. Since micro
scopic counting is often inherently fraught with
bias and individualism, a new test is being planned
which would subject the fluid nutrient of dispersed
HeLa cells suspended on the walls of test tubes
either to a photometric evaluation of a dye such
as neutral red (VP granules take up neutral red)
or to a counting method which would reveal the
radioactive material engulfed by the fluid droplets
and retained in the VP granules. These values
could then be related to the volume of packed
cells by a micro-hematocrit method (32). Such an
analysis would give essentially the same type of
information—i.e., the ratio of the number of VP
cells or, more specifically, the value which relates
the amount of VP granules to the total number
of cells (volume of packed cells). In such a test
many thousands of cells would be potential VP
FIG. 1.—Pinocytic cell of strain HeLa cultivated in the
multipurpose culture chamber in a basic fluid nutrient. In this
cell, the entering fluid droplets (arrows) rapidly diminished in
size and became the aggregated small black granules on the
lower and left side of the nucleus. Phase contrast, X900.
FIG. 2.—Pinocytic cell (VP cell) of strain HeLa cultivated
in the multipurpose culture chamber in a human serum (100
per cent) for 24 hours after initial cultivation for 7 days in a
basic fluid nutrient. In this cell the entering fluid droplets (ar
rows) apparently did not shrink but did display an index of re
fraction change and thus a phase reversal (white to black).
These now black and rounded fluid droplets (VP granules) ag
gregated in the cytoplasm and characterized the serum-pro
duced VP cell (variant pinocytic cell). Phase contrast, X900.
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ROSE—Strain HeLa Cell Response to Human Sera
cells, and, therefore, this enlargement of the cel
lular reaction for each serum analyzed should
increase the sensitivity of this type of biological
enumeration for cancer sero-diagnosis.
SUMMARY
1. An evaluation of a biological (tissue culture)
response by strain HeLa cells to 100 human sera
was presented.
2. This response was evaluated by a phase
contrast microscopic counting of the number of
VP cells formed in the periphery of HeLa colonies
(4-5-mm. diameter) after these colonies had been
cultivated for 24 hours (37°C.) in 100 per cent
human serum nutrients.
3. The response was higher in a greater per
centage of colonies cultivated in serum from cancer
patients and notably more so within a male group.
4. The high response within the male group was
shown to be due to the influence of the adenocarcinoma patients.
5. A method of refining this type of biological
sero-diagnosis was proposed.
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An Appraisal of a Biological Response by Strain HeLa Cells in
Tissue Culture to 100 Human Sera
George G. Rose
Cancer Res 1958;18:411-417.
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