Blood, Vol. 48, No. 2 (August), 1976 245

DNA Histogram Analysis of HumanHemopoietic Cells

By B. Barlogie, G. Spitzer, J. S. Hart, D. A. Johnston,

T. B#{252}chner,J. Schumann, and B. Drewinko

The proliferative activity of human neo-plasms r’isay be an important determinant

for therapeutic management. The adventof automated flow-through systems mea-sunng cellular DNA content by means offluorescence has considerably facilitatedthe analysis of cellular kinetics. Using apulse cytophotometer ICP-1 1 (Phywe Co.,G#{243}ttingen, Germany), three differentfluorescent staining techniques for DNAhistogram measurement on human hemo-poietic cells were tested: mithramycin,ethidium bromide, and a combination ofethidium bromide and mithramycin. Em-

ploying the tntiated thymidine labeling

index as reference standard for comparisonwith the DNA histogram-derived S-phasefractions, linear correlations were obtainedusing ethidium bromide alone and ethi-

dium bromide in combination with mithra-

mycin as staining techniques. The fluores-

cence intensity was increased fourfold to

fivefold by the use of the two-dye combin-

ation, resulting in a substantial decrease

in the coefficient of variation of DNA histo-

grams to 1 .5%-2%. This augmented his-

togram resolution is an important condi-tion for detecting small-degree numeric

chromosomal aberrations and discrete

drug perturbation effects.

C ONVINCING EVIDENCE has been accrued that, compared to normal

hemopoietic tissue, leukemic cells have a smaller growth fraction and a

longer generation time.’5 This finding is reflected by the lower labeling index of

leukemic bone marrow cells, as opposed to normal myeloid precursors.4 Re-

cently, several investigators have demonstrated the prognostic value of a pre-

treatment tritiated thymidine labeling index (LI) for subsequent response to in-

duction treatment.61#{176} In particular, Hart et al.� have shown, for three different

combination drug regimens for remission induction of previously untreated adultpatients with acute leukemia, that a blast LI > 9% is a favorable prognostic

factor independent of age. This conclusion is in keeping with observations in

tissue culture and in animal systems that most chemotherapeutic agents act

more effectively on rapidly proliferating cells. Attempts have been made to

manipulate the proliferative characteristics of the leukemic cell population by

in vivo drug perturbation. Thus, using a pulse dose of l-fl-D-arabinosylcytosine

(Ara-C) in patients with acute leukemia, Lampkin et al. have documented cell

synchronization and/or recruitment by triggering quiescent cells into the pro-

liferating compartment.’2 Such drug-induced increase of the LI may condition

patients presenting with a low LI for a better response to induction chemo-

From the Departments of Developmental Therapeutics. Biomathematics, and Laboratory Medicine.

the University of Texas System Cancer Center. M. D. A nderson Hospital and Tumor Institute.

Houston, Texas, and the University of M#{252}nster. M#{252}nster, Germany.

Submitted November 28, 1975; accepted April 19. 1976.

Supported in part by Grants CA -05831, CA-I 1520. and CA -14528 from the National Cancer In-

stitute.

Address for reprint requests: Barthel Barlogie. M.D., Department of Developmental Therapeutics.

University of Texas System Cancer Center. M. D. Anderson Hospital and Tumor Institute, 6723

Bertner A venue, Houston, Texas 77030.

1976 by Grune & Stratton. Inc.

For personal use only.on November 28, 2018. by guest www.bloodjournal.orgFrom

246 BARLOGIE ET AL.

therapy. This kinetic treatment approach requires frequent monitoring and

rapid availability of cell kinetic data, so that cytocidal chemotherapy can be

administered at the time of the peak LI of the leukemic population. With the

development of new in vitro culture systems for human hemopoietic cells,’3

analysis of differential kinetic and lethal response of leukemic and normal cells

to antineoplastic agents could possibly be used to spare normal stem cell dam-

age.

One of the reasons why cell kinetics has not yet been widely used in clinical

oncology is the laborious nature of previous technology. With the advent of

automated cytophotometry,’416 high-precision measurements of cellular com-

pounds such as DNA can be rapidly performed on a large number of cells.

Since DNA content is a function of cell cycle stage, DNA histograms allow a

more detailed analysis of the mitotic cycle, compared to techniques that mea-

sure the incorporation of radiolabeled nucleoside precursors into DNA.’7

Thus, cells in G,10, 5, and (G2 + M) phase can readily be identified. DNA

histography also allows detection of aneuploid abnormalities, independent of

the proliferative activity of the malignant cell clone,’8 whereas the capacity to

proliferate in vitro is an important determinant for the yield of metaphases in

routine cytogenetic techniques. However, such discrimination of aneuploid

clones on the basis of DNA content depends upon the resolution of the histo-

gram.

Recently, several flow-through cytophotometers have become commercially

available for the measurement of DNA content, and a variety of fluorescent

staining techniques have been reported.’932 Most of these techniques require

time-consuming procedures such as hydrolysis (Feulgen method and modifica-

tions) or RNase treatment, e.g., for ethidium bromide (EB) and propidium

iodide. Krishan has recently reported that by using propidium iodide in hypo-

tonic solution, rapid DNA histograms can be obtained which are identical to

those obtained after fixation and RNA digestion.28 Likewise, for mithramycin

(MI), which binds exclusively to double-stranded DNA, no preparative proce-

dures are required once monodispersed ceif populations have been generated.25

We have worked with a Phywe pulse cytophotometer ICP 1 1, which was de-

veloped by GOhde and Dittrich in l969.’� This instrument offers the advantage

that a number of fluorescent dyes can be used by altering the wavelength of

fluorescence excitation by means of different filters. For optimal excitation of

the MI-DNA complex, a wavelength of 390-400 nm is required. In a compara-

tive analysis of LI and DNA histogram-derived S-phase fractions of syn-

chronized cultures of human lymphoma cells, we have demonstrated the reli-

ability and accuracy of the MI staining technique for pulse cytophotometric

(PCP) analysis of this particular cell line.33

The purpose of this paper is twofold: (I) to compare different staining tech-

niques for DNA histogram analysis of hemopoietic cells, validating results

against the 3H-TdR LI; and (2) to present several examples of the utility of high

resolution DNA histography. Selecting the LI as reference standard for com-

parison with DNA histogram-derived S-phase compartment size, MI has

proved unreliable. A linear correlation between these two parameters was ob-

tained when hemopoietic cells were stained with EB alone or with EB and MI

For personal use only.on November 28, 2018. by guest www.bloodjournal.orgFrom

DNA HISTOGRAM 247

in combination. Compared to MI or EB alone, the two-dye technique led to a

substantial increase in fluorescence intensity, thus providing high resolution

DNA histograms with a coefficient of variation reduced to 1 .5#{176}�-2�/�. Em ploy-

ing these two fluorochromes in combination, we show that discrete compart-

ment changes in DNA histograms of hemopoietic cells can be detected. Thus,

drug perturbation effects and numeric chromosomal aberrations can be identi-

fled.

MATERIALS AND METHODS

Sample Processing

Bone marrow aspirates were obtained from 35 patients with acute leukemia using preservative-

free heparin as anticoagulant. The per cent blast cells ranged from 80% to l00#{176}/�.Immediately

after aspiration, the specimens were incubated with 3H-TdR (5 �iCi/ml; S.A. = 6.7 Ci/mM),

for 1 hr at 37’C. Erythrocytes were removed by Hypaque-Ficoll (HF) sedimentation (density =

1.078 g/cu cm, l000g for 15 mm at 4’C). Interphase cells were then collected�’35 and divided into

two aliquots for subsequent autoradiographic and PCP analysis. The LI was determined on 200

nucleated cells on cytocentrifuge preparations; cells with more than 5 grains overlying the nucleus

were considered labeled. For PCP analysis, the remaining aliquot ofthe HF-buffy coat was washed,

resuspended in 0.9% NaC1, and subsequently fixed in 70% ethanol.

Leukapheresed peripheral blood, and in three instances also bone marrow, from 15 patients

with leukemia were cultured in alpha medium supplemented with glutamine, L-asparagine,

L-serine, and 20#{176}/,fetal calf serum, at a cell density of 2 x l0� cells/mi in a 5% CO2 humidified

atmosphere at 37’C. In vitro proliferation was studied without exogenous stimulus, with 20%

leukocyte-conditioned medium, and in the presence of Difco-M PHA (0.005 ml per 2 x l0� cells).

DNA histogram and LI determinations were performed daily for 7 days. Cells were harvested

and incubated with 3H-TdR (5 MCi/mI; S.A. = 6.7 Ci/mM) for 60 mm at 37’C. One aliquot

of each sample was processed for autoradiography (cytocentrifuge preparation). The LI was

determined scoring 200 nucleated cells. For PCP, the remaining aliquot was washed and resus-

pended in 0.9% NaC1, and subsequently fixed in 70% ethanol.

Drugs

MI was kindly provided by Pfizer Labs, New York, N.Y. EB was obtained from Serva, Heidel-

berg, Germany. PHA was purchased from Difco, Detroit, Mich.; fetal calf serum from Gibco,

Santa Clara, Calif.; alpha medium from Flow Labs, Rockland, Md.

Staining Proceduresfor PCP Analysis

Ethanol-fixed cells were centrifuged at 1000 g for 2 mm and stained by one of the following

techniques: (1) 10 ml of MI 50 �g/ml with 7.5 mM MgCl2 and 12.5% ethanol (pH = 5.5) for

5 mm; (2) 10 ml of EB 25 pg/mI (in 0.1 M Tris buffer and 0.6% NaCI, pH = 7.4) for 15 mm, and

(3) a combination of EB and MI. The latter procedure involved staining cells initially in 5 ml of

EB 25 zg/ml(in 0.1 M Tris buffer with 0.6% NaCI, pH 7.4) for 10 mm; subsequently, 5 ml of Ml

50 g.g/ml (containing 7.5 mM MgCl2 and 12.5% ethanol) were added, resulting in final concen-

trations of 12.5 �g/ml of EB and 25 pg/mI of MI; after 5 more mm, the sample was measured in

the pulse cytophotometer. Routinely, 30,000-50,000 cells were measured for each DNA histogram.

Previous standardization experiments had shown that coincidences of fluorescent cells passing

simultaneously through the measuring chamber were negligible below a cell concentration of

5 x l0� cells/mi, generating less than 500 fluorescent signals/sec. When the pulse rate indicated

on the instrument exceeded 500/sec. the sample was further diluted with MI, EB, or an equivolu-

metric solution of the two-dye stock solution, respectively, so that EB = 12.5 pg/mI and MI =

25 pg/mI. Dye concentrations for MI, EB, and EB-MI were selected on the basis of previous

publications.2022’25’30’33

Unstained fixed cells could be stored at 4’C for 6 wk. For MI, EB, and EB-M I-stained cells, no

change in the histogram configuration was observed over a 2-hr period at room temperature. MI

For personal use only.on November 28, 2018. by guest www.bloodjournal.orgFrom

248 BARLOGIE ET AL.

0S% (PCP)

and EB-MI stained cells stored for 1-2 days at 4C showed no significant difference in DNA

histogram pattern with respect to that of freshly stained cells.

Treatment of ethanol-fixed cells (after rehydration) with RNase, 0.1%, for 30 mm at 37’C was

performed in some instances to test whether any ofthe three dye techniques also stained RNA.

Histogram Evaluation

DNA histogram evaluations of bone marrow samples and in vitro cultures were performed,

using the criteria of Baisch et al.36 for horizontal S-phase compartments. When the S-phase frac-

tion was skewed, a model suggested by Andreeff37 was applied, employing the mirror image

technique for G1 and (G2 + M) compartments. No significant difference between repeated mea-

surements of the same sample was observed; this confirmed the precision of the instrument.33

When split samples were measured, the standard deviation for the compartment distribution of

cells in G110, S, and (G2 + M ) fractions did not exceed 2%.

RESULTS

Technology

A comparative analysis of DNA histogram-derived S-phase fractions with in

vitro LI determinations was performed on bone marrow cells of eight patients

with acute leukemia, using MI as the DNA label (Fig. 1). Values scattered con-

siderably, with a multiple correlation coefficient (r) of 0.607 and an error mean

square (EMS) of 4.07. PCP-determined S-phase compartment size exceeded LI

values for the majority ofsamples. The coefficient of variation (CV) of the G110

peak of the DNA histograms ranged from 7% to 10%. When EB was used on

the remaining aliquots of the same fixed samples, a linear relationship was ob-

served with r = 0.994 and EMS = 1.2. Treatment with RNase 0.1% for 30 mm

at 37#{176}Cdid not change the histogram configuration, the compartment distribu-

tion, or the CV. The linear correlation between PCP-determined percentages

of S and LI was reconfirmed for the EB analysis by addition of nine more bone

marrow samples (r = 0.967, EMS = 1 .888), giving a CV of the G,,� compart-

ment of5%-lO% with a median of 7%.

The two-dye staining technique (EB-MI) was employed on another 18 con-

secutive bone marrow specimens. A marked increase in fluorescence intensity

and a decrease of the background fluorescence were observed. Thus, when the

pulse cytophotometer was adjusted so that the G110 peak of cells stained with

EB or MI alone appeared in channel 20, the G110 peak of EB-MI stained cells

was recorded in channels 85-90. This change reflected an increment in fluores-

cence intensity by a factor of four to five. Compared to each fluorochrome

Fig. 1. Correlation between U (3H-TdR) andS-phase fraction (PCP) of human bone marrow cellsfrom eight patients with acute leukemia, using EB orMl as fluorescent dyes for DNA histogram analysis.Processing techniques for autoradiography and PCPare described in the text. Samples were split afterethanol fixation and stained with EB 25 pg/mI (with

I 0.1 M Tris buffer and 0.6% NaCI, pH 7.4) and MI 50= 0994 pg/mI (with 7.5 mM MgCl2 and 12.5% ethanol), re-

EMS: 1.2 spectively. A linear correlation between percentages of

-j � p= 0.607 U and S (PCP) was obtained for EB as DNA labelEMS =4.07 (r= 0.994, EMS = 1.2). There was a broad scatter,

however, when MI was applied (r = 0.607, EMS =

4.07).

For personal use only.on November 28, 2018. by guest www.bloodjournal.orgFrom

r = 0.968EMS =2.634

S% (PCP)

50

40

30

20

I0

p =0969EMS =9#{149}4

I0 20 30

S% (PCP)

DNA HISTOGRAM 249

Fig. 2. Correlation between LI ( 3H-TdR)and S-phase fraction (PCP) of human bonemarrow cells from 1 8 patients with acuteleukemia, using EB and Ml in combination for

DNA histogram analysis. The staining pro-cedure of ethanol-fixed cells involved 10 mmstaining with EB 25 pg/mI (with 0.1 M Tris �buffer and 0.6% NaCI, pH 7.4); subsequently, �an equal volume of Ml 50 pg/mI (with 7.5 ,�

mM MgCl2 and 12.5% ethanol) was added,so that the final dye concentrations were EB, �.-

12.5 pg/mI and Ml, 25 pg/mI. After 5 mmmore, the sample was measured in the pulsecytophotometer ICP 1 1. A fourfold to five-fold increase in fluorescence intensity wasobserved, compared to either EB or MI alone.A linear correlation between percentages ofLI and S (PCP) was obtained (r = 0.968,

EMS = 2.634).

alone, the CV of the G,10 compartment was considerably improved, ranging

from 1.5% to 5%, with a median of 3%. As with EB alone, a linear correlation

between S-phase fraction and LI was obtained, with r = 0.968 and EMS =

2.634 (Fig. 2).

The EB-MI combination was further studied utilizing in vitro cultures of

leukemic peripheral blood and bone marrow (Fig. 3) with a total of 76 speci-

mens from 15 patients, harvested at different time intervals after initiation of

culture. A linear correlation between PCP-determined S-phase fraction and LI,

with a narrow distribution, was found (r = 0.969) in a fashion similar to that

of the analysis of fresh bone marrow specimens. In three instances, both MI

and EB-MI staining techniques were applied to split fractions of the same fixed

sample. Figures 4 and 5 show representative examples of the comparison be-

Fig. 3. Correlation between U (3H-TdR)and S-phase fraction (PCP) of hemopoieticcells in liquid culture, obtained from 15 pa-tients with leukemia. The culture conditionsare described in detail in the text. Altogether,

76 cultures (42 from peripheral blood and 34from bone marrow) were processed for auto-radiography and PCP. Therefore, cells were

harvested, incubated with 5 pCi/mI 3H-TdR

(S.A. = 6.7 Ci/mM) for 60 mm at 37’C. Onealiquot was further processed for auto-radiography (cytocentrifuge preparation); the

IT remainder was washed once with 0.9%NaCI, resuspended in 0.9% NaCI, and fixedwith ethanol for a final concentration of 70%.The fixed cells were stained with EB 25 pg/mI(with 0.1 mM Tris buffer and 0.6% NaCI,

pH 7.4) for 10 mm; subsequently, an equalvolume of Ml 50 pg/mI (with 7.5 mM MgCI2

and 12.5% ethanol) was added. After 5 mmmore, the sample was measured in the pulsecytophotometer ICP 11. There is a linear cor-relation between percentages of S (PCP) and

U(r=0.969,EMS = 9.4).

For personal use only.on November 28, 2018. by guest www.bloodjournal.orgFrom

cai PwI#{216}usr� Baood Liquid Cuftwsd2

(no stimuko)

s�m $ G2+M Cv

88% 10% 4% 2%

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tween DNA histograms obtained after MI and EB-MI staining techniques.

There was a significant difference in the CV of the G,10 peaks, with values of

2% and 5% in the case of EB-MI staining, compared to 5% and 8%, respec-

tively, for MI alone. In addition, compartment distributions differed to a con-

siderable extent in the example shown in Fig. 5. The simultaneously determined

LI in these two examples were in excellent agreement with the S-phase fraction

after EB-MI staining.

Figure 6 shows a series of DNA histograms (EB-MI combination technique)

AML - Sofa Mrrow

(HF . isquretion.fiaution in 70% ethanol)

$ G2+M CV

Mittiramycin 25 y�(mI +

thidium bromide

12.5 gig/mI 87% 9% 4% 5%

CIII � 36.000

- Mithr.mycin 50 gig/mI

C&l*78.000 81% 18% 1% 8%

0)C

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Channel Number

250 BARLOGIE ET AL.

Fig. 5. DNA histograms ofAML bone marrow in liquidculture, employing two differentstaining techniques: ER-MI incombination versus Ml alone.There is a difference for thecoefficient of variation (CV) ofthe G110 peak with a higherDNA histogram reselution incase of ER-MI. In addition, thecompartment distribution differsmarkedly. Since the U is 8.5%,the ER-MI technique appears tobe accurate.

Fig. 4. DNA histograms of CMLperipheral blood in liquid culture,employing two different stainingtechniques: ER-MI in combinationversus Ml alone. There is a differencefor the coefficient of variation (CV)of the G110 peak (channel 30), in-dicating a higher DNA histogramresolution for the two-dye stainingtechnique. The per cent compartmentdistribution of cells in G110, 5, and

(G2 + M) is different for the twostaining techniques. The LI was 10%.

For personal use only.on November 28, 2018. by guest www.bloodjournal.orgFrom

204060 204060 204060 204060 204060 204060

DNA HISTOGRAM 251

C

C

Channel No

- No Stimulus

Wi//i P1/4

Fig. 6. SequentIal DNA histograms of AML bone marrow cells in liquid culture: (A) withoutstimulus; and (B) with PHA 0.005 ml Difco-M/2 x 10� cells. In the absence of PHA, no significantchange in the histogram pattern can be observed. In the presence of PHA, however, a marked in-

crease of cells in 5- and (G2 + M)-phase compartments with a maximum on day 5 can be noticed.The G110 peak (channel 30) shows a broadening and asymmetry of the descending (right) slopeon day 4 of PHA culture; the peak of the 4C DNA complement mode is shifted beyond channel 60.

This phenomenon is no longer seen on day 5. (The cells were stained with ER and MI; 30,000.-50,000 cells were measured for each DNA histogram).

ofacute myeloblastic leukemia (AML) bone marrow cells harvested between

days 1 and 7 of culture, both with and without PHA. An asymmetry of the

G,10 peak with skewness of the descending (right) slope could be noticed on

the fourth day of the stimulated culture. The (G2 + M) peak was shifted be-

yond channel 60. This effect could no longer be seen on day 5. Since we did not

use reference cells,33 the ploidy of the major peak could not be identified. The

instrument was, therefore, adjusted so that the G,10 peak appeared in channel

30.

RNase treatment applied to eight samples before staining (four for each

staining technique) did not result in alterations ofthe compartment distribution

ofcells in G,,�, S, and (G2 + M) phase or the histogram resolution.

Clinical Application

In the following part, we present some preliminary observations, which illus-

trate the potential application of DNA histography to clinical problems. The

high resolution EB-MI staining technique was employed in these studies.

Figure 7 shows a series of DNA histograms of both bone marrow and pe-

ripheral blood of a patient with poorly differentiated lymphocytic lymphoma.

On presentation, 88% lymphoma cells were detected in the peripheral bloodwith a mitotic index of 0.1%. Interestingly, on DNA histogram analysis (Fig.

7A), the majority of the peripheral blood cells (84%) were in the 4C DNA

complement range of(G2 + M) cells. Similarly, the bone marrow with a lesser

infiltrate of lymphoma cells (3 5%) also showed an increase of the (G2 + M)compartment (33%). Routine cytogenetic screening38 detected only two cells

that had a diploid karyotype. However, employing the technique of premature

For personal use only.on November 28, 2018. by guest www.bloodjournal.orgFrom

A

BloodBone Marrow92575

(prior to treatment)

35% lymphomocells with LI:O%

t33� A

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�30 602C 4C

30 60 Channel Na2C 4C DNA Complement

252 BARLOGIE ET AL.

88% /yinphomo Fig. 7. Sequential DNA

ce//s wiThLl:/% histograms of bone marrowand blood of a patient withmalignant lymphoma in Ieu-kemic phase (poorly dif-ferentiated lymphocytic type).Ethanol-fixed cells were stained

__________ __________�84% with ER and Ml; 30,000-50,000 cells were measuredfor each DNA histogram. Theslashed areas approximatethe tetraploid lymphoma cellpepulation. Notice the closecorrelation between the percent tetraploici cells (PCP) and

the fraction of lymphoma cellsidentified morphologically on

____________ May.-G r#{252}nwald-Giem sa-stained smears. After 3 mo ofintensive combination chemo-therapy, the bone marrownearly cleared of lymphomacells, whereas the peripheralblood still contained a con-siderable fraction of malignantcells. The increase of lym-phoma cells in the bone mar-

row in (B) may be due tocontamination with peripheralblood.

chromosomal condensation (PCC),39 which can visualize chromosomal ab-

normalities of interphase cells, we were able to document a tetraploid karyo-

type. Further specimens obtained during the subsequent management of this

patient showed a close correlation between the per cent lymphoma cells in both

peripheral blood and bone marrow, and the per cent cells in (G2 + M) on

DNA histograms (Fig. 7A-C).

Figure 8 shows a series of DNA histograms of bone marrow and blood cells

of a patient with acute myelogenous leukemia. Two peaks in the G110 range of

the DNA histogram were observed. To identify the ploidy of these peaks,

diploid chronic lymphocytic leukemia cells were mixed with this patient’s

samples prior to staining with EB and MI in combination. The right peak in

both bone marrow and blood of this patient coincided with the diploid peak

(not shown). Again, cytogenetic analysis revealed a small number of only

diploid karyotypes, but no aneuploidy. However, PCC analysis documented

the presence of cells with 44 chromosomes. Ultrastructural examination

demonstrated the presence of nuclear blebs in the blast cells. These blebs have

been reported previously to be associated with leukemic cytogenetic abnor-

malities.4#{176} Residual normal myeloid progenitor cells were detected by in vitro

agar culture.4’

Recently, we have initiated a study designed to analyze the incidence and

time course of cell synchronization and/or recruitment induced by a single

For personal use only.on November 28, 2018. by guest www.bloodjournal.orgFrom

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Bone Marrow Blood

8efore 4R4�C’X/0mg/m2LVpush

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x13O x2 602C 4C

x 30 x2 60 Channel No.X 4C DNA Complement

DNA HISTOGRAM 253

Fig. 8. SequentIal DNA histograms ________of bone marrow and peripheral bloodof a patient with AML before and afteran i.v. push injection of Am-C 200mg/sq m. Ethanol-fixed cells werestained with ER and Ml; 30,000-50,000 cells were measured for eachDNA histogram. To identify the ploidy .�

of the two peaks in the 2C range �of the histograms, diploid chronic ________________lymphocytic leukemia (CU.) cells wereadmixed to aliquots of the patient’s �

samples prior to the staining proce- �dure. In the resulting DNA histograms(not shown), the right peak in both

bone marrow and blood coincidedwith the diploid (CU.) peak. Conse-quently, the slashed area approxi-mates a hypodiploid leukemic blast ________________cell population with a G110 peak atxl and a (G2+M) peak at X2. Thefraction of these cells with an ab-

normal DNA content correlates with

the percentage of blast cells onmorphological examination. The peak

in channel 30 represents the residualnormal diploid pepulatlon. Notice the

consistency of the histogram configura -________Hon over a 3-day period with distinctpeaks at X1 and in channel 30.

intravenous push dose ofAra-C 200 mg/sq m.’2 Figure 9 shows a series of bone

marrow DNA histograms of a patient with acute undifferentiated leukemia

(> 95% blast cells) prior to and 4, 24, and 72 hr after a pulse dose of Ara-C.

Compared to the pretreatment compartment distribution, the (G2 + M ) phasefraction had decreased at 4 hr (from 2% to < 1%). Twenty-four hours after

administration of Ara-C, a cohort of 40% of cells was seen in early S phase,

with a skewness of the lower descending (right) slope of the G,10 peak. At 72 hr,

the S-phase compartment showed a configuration with 20% cells equally dis-

tributed between the 2C and 4C DNA complement.

DISCUSSION

The S-phase fraction of the DNA histogram is defined as a cohort of cells

with a DNA content higher than the 2C DNA complement of diploid G,10 cells

and less than the 4C DNA complement of (G2 + M) phase cells. The tritiated

thymidine LI reflects the percentage of cells synthesizing DNA.42 Thus, both

the PCP-determined S-phase fraction and percentage of LI should be identical

For personal use only.on November 28, 2018. by guest www.bloodjournal.orgFrom

A

083%

5 :/5%

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under unperturbed conditions. We have evaluated three different DNA staining

techniques for their usefulness in obtaining DNA histograms of human hemo-

poietic cells, and have tested the validity of this determination by using the

3H-TdR LI as the reference value ofS-phase cells.

MI is a DNA-specific fluorescent dye that complexes with native DNA. Al-

though the exact mechanism of dye-DNA interaction has not yet been eluci-

dated,32’43� there is evidence of base specificity for guanine, and Mg2� ions are

required in an equimolar concentration to mithramycin for maximum interac-

tion. Our previous study utilizing a lymphoid cell line has shown a close cor-

respondence between LI and PCP-determined S-phase fraction.33 When applied

to hemopoietic human cells, a marked discrepancy between DNA histogram-

derived S-phase fractions and simultaneously determined LI is found. Thus,

under the conditions described, this dye does not meet the criteria which we

have chosen to assess the validity of DNA-staining techniques.

EB is an intercalating agent, which preferentially binds to double-stranded

DNA, but also to RNA.29 In this study, a good agreement between LI and

254 BARLOGIE ET AL.

Fig. 9. Sequential DNA histograms of bonemarrow cells of a patient with AML before (A)and 4 hr (B), 24 hr (C), and 72 hr (D) after a

single i.v. push injection of Am-C 200 mg/sq m.(B) There is a disappearance of late S and (G2 +M) cells at 4 hr. (C) There is a large cohort of cells(40%) in early S phase after 24 hr. (D) After 72 hrthe cells are equally distributed throughout Sphase, as indicated by the horizontal part of the

DNA Complement histogram curve.

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DNA HISTOGRAM 255

PCP-determined S-phase fraction values has been observed, even without the

use of RNase treatment.

When EB and MI were used in combination, a fourfold to fivefold increase in

fluorescence intensity and a decrease of the background fluorescence was noted.

Thus the DNA histogram resolution was substantially improved and the CVs

of the G,10 peak were reduced to minimum values of 1.5%. The mechanism

by which augmentation of fluorescence emission occurred is not clear at

present. The maximum level of fluorochrome binding is 0.1 molecules per

nucleotide for MI and 0.25 for EB.� Assuming different binding sites for

the two fluorochromes, the increase in fluorescence intensity may be due to

additive binding. Also, energy transfer may be involved, as has been suggested

for the combination of EB and Hoechst 33258 by Berkhan.2’ The sequential

staining process (EB followed by MI) rendered slightly higher resolutions com-

pared to either simultaneous or inverse sequence. Additional RNase treatment

did not improve the quality of the DNA histograms. There was excellent cor-

relation (r = 0.968 for bone marrow samples and r = 0.969 for cultured cells)

in the regression analysis of LI and S-phase percentage, documenting the

validity of the combination staining technique. The total processing time (15

mm for HF separation and 15 mm for staining) exceeds the 5 mm reported by

Krishan for his rapid propidium iodide staining technique.28 However, this

minor time delay disadvantage is amply offset by the increased resolution of the

DNA histogram attained by EB-MI staining. This consistently high DNA

histogram resolution is an important factor in the detection ofminor changes in

compartment size distribution.

A peculiar change in the histogram pattern was observed on day 4 of one

PHA-stimulated culture and consisted ofbroadening and asymmetry ofthe G,10

peak. This effect may be due to chromatin decondensation occurring in cells

entering the proliferative cycle out of G0, thus binding more fluorochrome.

Such increased binding has also been observed for acridin orange45 and EB.�

Chromosomal aberrations have been found in 40%-50% of patients with

acute leukemia,38’47 and may be associated with a poorer response to chemo-

therapy.38 They are useful to monitor residual or relapsing disease. Detection

of karyotype abnormality by routine cytogenetic techniques depends on the

proliferative potential of the abnormal cell clone. We have presented two ex-

amples of DNA histogram abnormalities, one with a large fraction of cells in

the tetraploid (G2 + M) range and another with an extra peak in the hypo-

diploid range. Our suspicion of an abnormal karyotype in the absence of

chromosomal aberration on routine cytogenetic screening was supported by

PCC analysis and the finding of nuclear blebs in the second patient. Growth of

normal myeloid progenitor cells in agar culture suggests that the normal cyto-

genetic karyotype and cells with a 2C DNA complement (PCP) may be derived

from residual normal hemopoietic cells. In both examples, the fraction of cells

with abnormal DNA content was in close agreement with the percentage of

morphologically abnormal-appearing cells. Detectability of chromosomal ab-

normality by measurement of DNA content is independent of the kinetic

behavior of the cells, but is determined by the difference in DNA content com-

pared to normal diploid cells. Therefore, high-resolution histograms are man-

datory for discrimination of small degree numeric chromosomal aberrations.

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256 BARLOGIE ET AL.

The example of drug perturbation with Ara-C revealed an increment of cells

in the early S-phase range. The detection of such drug-induced changes again

depends entirely on the difference in DNA content of the perturbed cells in

relation to the histogram resolution. In case ofa low CV, compartment changes

in the G,10-S and S-(G2 + M) range may escape recognition.

PCP-determined DNA histograms employing EB and MI in combination can

provide fast information on fluctuations of discrete compartments of the cell

cycle and, thus, offer the rationale for pulses of chemotherapy scheduled on the

basis of rapidly available kinetic characteristics. Since DNA histograms do not

allow cell identification on a basis other than DNA content, results obtained

for mixed cell populations must be interpreted with caution. Advances in cell

separation methodology should enable us to analyze rapidly and sensitively cell

cycle changes in both leukemic and residual normal hemopoietic cells induced

by chemotherapy.485#{176} Thus, differences in kinetic response of normal and leu-

kemic cells to antineoplastic agents may be advantageously exploited to in-

crease the lethal effects on malignant cells, while sparing normal host stem cells.

ACKNOWLEDGMENT

We gratefully acknowledge the technical skills and assistance of Ms. Susan Sumners and Ms.

Marcia Lomedico. The authors wish to express their gratitude to Dr. M. J. Ahearn, who performed

the ultrastructural investigations, and to Dr. W. Hittelman, who carried out the PCC analyses.

We appreciate the secretarial assistance of Ms. Diane Teltschik.

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1976 48: 245-258  

B Barlogie, G Spitzer, JS Hart, DA Johnston, T Buchner, J Schumann and B Drewinko DNA histogram analysis of human hemopoietic cells 

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